Polynucleotides Encoding Proteins with Clot-Specific Streptokinase Activity

ABSTRACT

The present invention provides polynucleotides encoding clot-specific streptokinase proteins possessing altered plasminogen characteristics, including enhanced fibrin selectivity. The kinetics of plasminogen activation by these proteins are distinct from those of natural streptokinase, in that there is a temporary delay or lag in the initial rate of catalytic conversion of plasminogen to plasmin. Also disclosed are processes for preparing the proteins.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.10/631,558, filed 31 Jul. 2003, which is a divisional of U.S.application Ser. No. 09/940,235, filed 27 Aug. 2001, now U.S. Pat. No.7,163,817, which is a continuation of U.S. application Ser. No.09/471,349, filed 23 Dec. 1999, which claims priority from IndianApplication No. 3825/DEL/98, filed 24 Dec. 1998. The disclosure of theprior applications are considered part of (and are incorporated byreference in) the disclosure of this application.

FIELD OF THE INVENTION

The present invention relates to novel clot specific streptokinaseproteins possessing altered plasminogen characteristics. The inventionfurther relates to a process for the preparation of the said proteins.The streptokinases so produced have properties of chanced fibrinselectivity as well as kinetics of plasminogen activation that aredistinct from that of natural streptokinase in being characterized by atemporary delay, or lag, of several minutes in the initial rate of thecatalytic conversion of plasminogen to plasmin (a process termedhereafter as PG activation”).

The advantage of this invention lies in the presence of these twoproperties in these chimeric (or hybrid) proteins together i.e. fibrinaffinity and an initial lag in plasminogen activation. In other words,the hybrid protein molecules disclosed in this invention are both fibrinspecific and display “delayed-action” thrombolysis. This confers onthese novel proteins the ability to bind tightly with fibrin, theproteinaceous substance of blood clots soon after their introductioninto the vascular system without significantly activating thecirculating blood plasminogen to plasmin, thus aiding in thelocalization of the PG activation process to the site of thepathological thrombus. Thus, once the PG activation lag is overcomewithin a few minutes' of the exposure of the hybrid proteins toplasminogen, they can easily activate the plasminogen in the immediatevicinity of the thrombus in a manner essentially similar to that ofnatural i.e. unmodified streptokinase, thereby obviating the systemic PGactivation frequently encountered during clinical use of streptokinase.These new hybrid proteins can therefore be used to advantage forthrombolytic therapy for various kinds of cardiovascular disorders.

BACKGROUND OF THE INVENTION

In recent years, thrombolytic therapy with fibrinolytic agents, such asStreptokinase (SK), tissue plasminogen activator (TPA) or urokinase (UK)has revolutionized the clinical management of diverse circulatorydiseases e.g., deep-vein thrombosis, pulmonary embolism and myocardialinfarction. These agents exert their fibrinolytic effects throughactivation of plasminogen (PG) in the circulation by cleavage of thescissile peptide bond between residues 561 and 562 in PG. As a result,the inactive zymogen is transformed to its active form, the serineprotease, plasmin (PN), which then acts on fibrin to degrade the latterinto soluble degradation products. It may be mentioned here that PN, byitself, is incapable of activating PG to PN; this reaction is catalyzedby highly specific proteases like TPA, the SK-plasminogen complex, andUK, all of which possess an unusually narrow protein substratepreference, namely a propensity to cleave the scissile peptide bond inPG. However, unlike UK and TPA, SK has no proteolytic activity of itsown, and it activates PG to PN indirectly by first forming ahigh-affinity equimolar complex with PG, known as the activator complex(reviewed in Castellino, F. J., 1981, Chem. Rev. 81: 431).

Of the several thrombolytic agents used clinically, SK is probably oneof the most-often employed, particularly because of its markedly lowercost when compared to TPA and UK. However, the choice of thrombolyticagent during therapy is dictated by a number of factors besides cost,such as the presence of side-effects and their severity, in vivometabolic survival of the drug (e.g., circulatory clearance rates),fibrin selectivity and/or affinity, immunological reactivity etc. SK isa highly potent PG activator, and has a relatively long plasmahalf-life—properties that, together, impart a certain advantage to thisdrug as compared to its counterparts viz, TPA and UK. However, due to alack of any appreciable fibrin clot-specificity in the former, theadministration of therapeutically effective doses of SK often results insystemic PG activation, resulting in hemorrhagic complications due tothe proteolytic degradation of blood factors by the plasmin generatedthroughout the circulatory system. However, if a fibrin affinity and/orselectivity could be integrated into SK a molecule which otherwisepossesses little fibrin affinity of its own, it would considerablyenhance the therapeutic efficacy of this thrombolytic agent. Withrespect to the other covered trait in a fibrinolytic agent, such as thatdescribed above for TPA above (viz., considerably lowered activity whilecirculating in the vascular system but enhanced PG activating ability inthe presence of fibrin), attempts have been made in the past to produceanalogs of SK with greater circulatory half-lives and decreased systemicplasmin generation by incorporating properties such as a slower rate ofPG activation into the fibrinolytic agent. One example where this hasbeen successfully accomplished is that of anisoylated streptokinaseplasmin activator complex, abbreviated APSAC (sold under the trade-name‘Eminase’ by the Beecham pharmaceutical group) (reference: Smith, R. A.G., Dupe, R. J., English, P. D., and Green, J., 1981, Nature 290:505) inwhich the catalytically important serine residue of the plasmincomponent is blocked by reversible acylation. The generalized plasminactivation coincident with the administration of unmodified SK has beenreported to be appreciably diminished during thrombolytic therapy withAPSAC since the deacylation of the covalently modified serine in theSK-acylated plasmin complex occurs slowly in the vascular system.

It is thus generally recognised that it will be of significant clinicaladvantage if SK could be engineered to possess increased fibrinaffinity/specificity together with a markedly slower initial rate ofactivation of PG. Thus, soon after injection into the body, whilst it isstill in an inactive or partially active state, such a modified PGactivator will bind to the pathological fibrin clot during its initialsojourn through the vascular system in an inactive/partially activestate. However, after an initial lag (a property engineered into thederivative/analog through design) it will become fully activated afterbeing sequestered to the fibrin clot by virtue of its fibrin affinity.Thus, the PG activation process will be relatively limited to theimmediate vicinity of the clot, thus obviating the systemic PGactivation coincident with natural SK administration which has nointrinsic fibrin affinity of its own and which activates PG as soon asit encounters it. In other words, whilst the former property in thenovel protein/s would be expected to confer on the thrombolytic agent anability to target itself to the immediate locale of the pathologicalclot and thus help build up therapeutically effective concentrations ofthe activator therein, the initially slow kinetics of PG activationwould result in an overall diminished generation of free plasmin in thecirculation. The net result hall be a continued and more efficientfibrinolysis at the target sustained by considerably loweredtherapeutically effective dosages of the thrombolytic agent.

In the past, the gene encoding for SK has been isolated from its naturalsource (Streptococcus species) and cloned into several heterologousmicro-organisms such as yeast (Hagenson, M. J., Holden, K. A., Parker,K. A., Wood, P. J., Cruze. J. A., Fuke. M., Hopkins, T. R., Stroman, D.W., 1989, Enzyme. Microb. Technol. 11:650), bacteria viz, E. coli(Malke, H, Ferretti, J. J., 1984, Proc. Nat'l. Acad. Sci. 81: 3557),alternate species of Streptococcus (Malke, H., Gerlach, D., Kohler, W.,Ferretti, J. J., 1984, Mol. Gen. Genet. 196:360), and Bacillus (Wong, S.L., Ye, R., Nathoo S., 1994, Applied and Env. Microbiol. 1:517). Inaddition, genetically modified SK derivatives containing “Kringle” typefibrin binding domains derived from plasminogen, and methods ofobtaining the same by rDNA techniques, have been described (EU 0397 366A1). However, since five such Kringl regions are already present in thenatural SK-PG activator complex, being an integral part of PG in theactivator complex, the advantages gained from further addition of suchdomains art likely to be minimal. Hence, there is a need to impart aqualitatively different fibrin-affinity and/or specificity to theactivator complex, particularly of a type associated with TPA, a veryeffective thrombolytic agent possessing much greater fibrin affinitythan SK. TPA is known to contain a fibrin-associating “finger” domain,which is structurally and functionally very similar to thefibrin-binding domains present in fibronectin, a multi-functionalprotein with ability to interact with a number of other proteins besidesfibrin e.g., collagen, heparin, actin etc (reviewed in Ruoslahti. E.,1988, Ann. Rev. Biochem. 57:375). Methods for the imaging offibrin-containing substances, such as pathological olots and/oratherosclerotic plaques in vivo by using large radio-labeledpolypeptides derived from fibronectin, and gearing these FBDs (fibrinbinding domains) have been disclosed (see: PCT WO 91/17765); this patentalso discloses chemically cross-linked FBD-containing polypeptides and athrombolytic agent (SK) to effect thrombus-targeted fibrinolysis. Thechemical cross-linking procedure resulted in the generation of a complexmixture of heterogeneously cross-linked molecules with variable FED andSK content, since the bifunctional agents used for chemicalcross-linking essentially cross-link any of the large number of lysineside-chains present in the participating molecules viz SK and HPG. Thus,this procedure generates mixtures of molecules with undefined locationof the cross-links between the molecules e.g. both dimers and multimerswith both hom—(e.g. SK-SK or FBD-FBD types) and hetero-crosslinkedmolecules with varying sites of cross-links are expected to be formed.In addition, it is noteworthy that the SK molecules chemicallycross-linked with fibrin binding polypeptides disclosed in this patentshowed an overall level of PG activator activity essentially comparableto that of unmodified SK, and no alteration was observed in the rate ofPG activation, or the presence of an initial lag in the PG activationkinetics. It is quite clear that this invention related to thepreparation of a heterogeneous population of cross-linked molecules withstructures essentially undefined with respect to the cross-links'locations, and without any cross-correlation between the differentstructures in the ensemble of molecules and their correspondingfunctional properties. This is a serious limitation in the descriptionof a drug intended for therapeutic application, in general, and withrespect to the exact nature of the structure-function correlation in thecollection of the cross-linked molecules, in particular.

In the past, hybrid SK derivatives with “kringle” type fibrin bindingdomains derived from human plasmin(ogen) fused to the former, andmethods of obtaining the same by rDNA techniques, have been described(EU 0397 366 A1 and U.S. Pat. No. 5,187,098). However, five such Kringleregions are already present in the natural SK-Plasmin(ogen) activatorcomplex, as noted before, being an integral part of PG in the activatorcomplex, which has a weak fibrin affinity at best (Fears, R., 1989.,Biochem. J. 261: 313). Hence, there is a need to impart a qualitativelydifferent fibrin-affinity and/or specificity to the activator complexand utilize the affinity so imparted to obtain SK derivatives thatdisplay functional characteristics that help avoid the immediateactivation of plasminogen upon contact with the latter.

Certain proteins are known to contain fibrin-associating “finger”domain/s, such as those present in fibronectin, a multi-functionalprotein with ability to interact with a number of other proteins besidesfibrin e.g., collagen, heparin, actin etc (reviewed in Ruoslahti, E.,1988, Ann. Rev. Biochem. 57:375). TPA also possesses a “finger” typefibrin binding domain (FBD) that greatly helps in its fibrin association(Verheijen, J. H. et al., 1986., EMBO J.vol. 5, pp. 3525). Methods forthe imaging of fibrin-containing substances, such as pathological clotsand/or atherosclerotic plaques in vivo by using large radio-labeledpolypeptides derived from fibronectin, and bearing these FBDs have beendisclosed (see, PCT WO 91/17765); this patent also discloses chemicallycross-linked FBD-containing polypeptides and a thrombolytic agent (SK)to effect thrombus-targeted fibrinolysis. However, it is noteworthy thatthe SK molecules chemically cross-linked with fibrin bindingpolypeptides showed an overall level of PG activator activityessentially comparable to that of unmodified KS, and no alteration wasobserved in the rate of PG activation or the presence of an initial lagin the PG activation kinetics. Besides, the cross-linking procedureresulted in the generation of a complex mixture of heterogeneouslycross-linked molecules with variable FBD and SK content, since thebifunctional agents essentially cross-linked any of the large number oflysine side-chains present in the participating molecules viz. SK andHPG likely generating both dimers and multimers with both homo- (e.g.,SK-SK or FBD-FBD types) and hetero-crosslinked molecules. Moreover, thisinvention essentially disclosed the preparation of a heterogeneouspopulation of chimeric molecules between SK and fibrin bindingpolypeptide with undefined covalent structures with respect to the sitesof cross-linking well as types of polymers so formed i.e. whether homo-(SK-SK or FBD-FBD types) or hetero-types, so that any meaningfulstructure-functional cross-correlation between the different structuresin the ensemble and their corresponding functional properties cannot beobtained. This is a serious limitation in a drug intended fortherapeutic application particularly one administered through aparenteral route in human beings.

In contrast the present invention provides novel clot-specificstreptokinase proteins possessing altered plasminogen activationcharacteristics and a process for the preparation of different types ofsaid proteins by recombinant DNA technology which have been designedusing precisely defined elements of DNA polynucleotides that encode forfibrin binding domains and SK, or their modified forms. The hybridproteins so formed thus have two very important structural as well asfunctional elements, namely SK or its modified forms, and finger typefibrin binding domain/s attached to each other through covalent peptidebonds in a predefined and predetermined order of juxtaposition withrespect to each other (see FIG. 1 for types of such constructs, and therationale for their construction, which is provided below) so that thehybrid, or chimeric, proteins so produced after expression in a suitablesystem possess discrete, definable covalent structures. In other words,the novel hybrid proteins contain SK or functionally relevant partsthereof, connected through polypeptide linkage/s with the relevantprotein domains of human fibronectin that are capable of independentlyconferring fibrin affinity to the resultant hybrids in such a mannerthat the hybrid protein/s specifically display altered plasminogenactivation characteristics. The latter is marked by the presence of aninitial period of lag of several minutes' duration in the rate of PGactivation by the hybrid SK derivatives (viz., time-delayed PGactivation), which is followed by high rates of PG activation akin tothat displayed by unmodified SK. In other words, the duration of theinitial lag, which varies depending on the type of hybrid construct, israpidly followed by PC activation rates closely similar to that ofnatural type SK. The simultaneous presence of the afore-mentioned twodistinct biochemical properties in the same cot-dissolver proteinmolecule renders these hybrid streptokinases as very useful drugs fortargeted, time-delayed clot lysis during thrombolytic therapy.

The biologically active form of Streptokinase (SK) is either theSK-plasminogen or SK-plasmin molecule/s, formed in the circulatorysystem by the association of SK with endogenous plasminogen soon afterits administration in vivo. This complex is also known as the activatorcomplex, a highly specific protease that activates substrate moleculesof plasminogen to plasmin, which proteolytically digests fibrin andhelps restore blood circulation in occluded vessels (Castellino, C. J.,1981., Chem. Rev. 81:431). Unlike free SK, which does not possess fibrinaffinity, this complex already possesses substantial fibrin affinity ofits own due to the “kringle” fibrin binding domains present in theplasmin(ogen) part of the SK-plasmin(ogen) activator complex Fears R.,1989., Biochem. J. 261:313 see also references cited therein).Nevertheless, unlike other preferred plasminogen activator protein drugssuch as tissue plasminogen activator (TPA) which possesses intrinsicfibrin affinity as well as a fibrin-dependent plasminogen activationkinetics, the administration of SK during clot dissolution therapy oftenleads to unwanted systemic activation of plasminogen throughout thecirculatory system due to immediate activation of circulatingplasminogen, as opposed to the desired activation in and around thefibrin clot occluding the flow of blood through the affected vessel/s.

Thus, it will be of significant clinical advantage if SK could beengineered to possess increased fibrin affinity/specificity togetherwith a markedly slower initial rate of activation of plasminogen (PG),but becoming capable of activating plasminogen in a manner similar tothat of unmodified SK after an initial hiatus. Thus, soon afterinjection into the body, whilst it is still in an inactive or partiallyactive state, the engineered SK will bind to the pathological fibrinclot while still in an inactive or partially active state, as itsojourns through the vascular system by virtue of the engineered fibrinaffinity. However, after the initial lag in its PG activation kineticsis overcome in a few minutes, it will preferentially become activated inthe immediate vicinity of the clot where it is now sequestered, therebyobviating or significantly minimizing the systemic PG activationcoincident with natural SK administration which immediately activates PGupon administration. Thus, whilst the former property (of fibrinaffinity) would be expected to confer on the new thrombolytic agent anability to target itself to the immediate locale of the pathologicalclot and thus help build up therapeutically effective concentrations ofthe activator therein, the other property (of an initially slow kineticsof PG activation) would result in an overall diminished generation offree plasmin in the circulation. The net result shall be a continued andmore efficient fibrinolysis at the target sustained by considerablylowered therapeutically effective dosages of the thrombolytic agent. Inconclusion, a fibrin affinity per se in SK has little beneficialconsequences (which anyway the SK-PG complex possesses in some measure)unless the systemic PG activation is thwarted and/or delayed.

An important attribute of the present invention is the preparation ofdifferent types of novel and hitherto undisclosed chimeric SKderivatives produced by recombinant DNA technology using definedgene-segments of SK and FBD combined in a pre-designed manner. Thesenovel genetic constructs have been designed using precisely defined DNAelements that encode for SK and fibrin binding domains, or theirmodified forms so as to retain the functional characteristic of each (PGactivation and fibrin affinity, respectively) as well as acharacteristically altered PG activation kinetics. The chimeric proteinsso produced have two types of elements (SK and the ‘finger’-type fibrinbinding domains, or their modified forms) in a predefined andpredetermined order of juxtaposition with respect to each other, so thatthe chimeric proteins expressed from these genes possess discrete,definable covalent structures. In other words, the chimeric proteinscontain SK or parts thereof, connected through polypeptide linkage withthe relevant protein domains that confer fibrin affinity to theresultant hybrids and also specifically result in altered kinetics of PGactivation. The latter is characterized by an initial lag, or absence ofPG activation, of several minutes' duration (viz., time-delayed PGactivation), followed by high rates of PG activation akin to that ofunmodified SK. The initial lag (which varies from approx. 8 min to 25min depending on the design of the SK derivative) is rapidly followed byhigh rates of PG activation closely similar to that displayed by naturaltype SK. The simultaneous presence of the afore-mentioned twobiochemical properties in the same PG activator molecule has not beendisclosed in the SK-derived molecules in either of the patentdisclosures cited above. In addition, the present patent discloses newcombinations of DNA sequences that have been used to express the novelprotein molecules with a unique combination of functional properties,mentioned above, which are not disclosed in the other patents.

The rationale for the construction of hybrid SK derivatives as disclosedin the process of the present invention with both fibrin specificity anddelayed PG activation kinetics is explained below.

The molecular basis for the fibrin affinity displayed by fibronectin hasbeen studied in some detail in recent years (Matsuka, Y. V., Medved, L.V., Brew. S. A. and Ingham; K. C., 1994, J. Biol. Chem. 269:9539). Underphysiological conditions, FN first interacts reversibly (but withrelatively high affinity) with fibrin and is then covalentlyincorporated into the fibrin clot matrix through clotting factor XIII, atransglutaminase (reviewed in Ruoslahti, E., 1988, Anna. Rev. Biochem.57:375), whose action results in the covalent cross-linking between FNand a lys residue in fibrin(ogen) at the reactive Gln (residue 3) of theformer. The region/s responsible for the interaction of FN with fibrinhave been identified to resin both in the N-terminal as well as theC-terminal ends of this multi-domain protein. The N-terminal region ofFN comprises of five finger modules (FBDs) as well as a transglutaminasecross-linking (TG) site, whereas the C-terminal region, lacking a TGsite, contains three modules, as demonstrated by the binding ofdifferent polypeptides derived from FN carrying these two broad regionsto fibrin-agarose. The exact domains in the N-terminal regionresponsible for the strong binding of the FN module, and their relativecontributions towards this interaction have been analysed closely(Matsuka, Y. V., Medved, L. V., Brew, S. A. and Ingham, K. C., 1994, J.Biol. Chem. 269:9539 and Rostagno et al., 1994; J. Biol. Chem. 269,31938) by expressing DNA segments encoding various combinations of themodules in heterologous cells and/or by examining the fibrin bindingproperties of polypeptide fragments carrying these modules prepared bylimited proteolysis of FN. These studies clearly identified that of allthe individual modules present in the N-terminal region of FN, thebi-modular arrangement viz., FBD 4 and 5 domains, displayed a fibrinaffinity significantly comparable to the interaction of the full-lengthFN molecule, in contrast to all the other domains either as pairs orindividually (including 4 and 5) which displayed poor affinity at 37° C.It is therefore clear from these studies that physiologically effectivefibrin binding is not a common property of all the modules, eitherindividually or in pairs, but is principally located in the FBD pair of4 and 5, and to a relatively lesser extent, in domains 1 and 2.

To achieve the functional objective of an initially time-delayed PGactivation kinetics by the hybrid SK derivatives, our design utilizesthe fusion of selected regions of the FBDs of human fibronectin or itshomologous sequences present in other proteins with SK (or its partiallytruncated forms) at strategically useful points so as to kineticallyhinder the initial interaction of SK with PG necessary to form the 1:1stoichiometric activator complex. It is known that of the 414 residuesconstituting native SK, only the first 15 residues and the last 31residues are expendable, with the resultant truncated polypeptide beingnearly as active as the native full-length protein in terms of PGactivation ability. Further truncation at either end results in drasticdecrease in the activity associated with the molecule (Malke, H., Roe,B., and Ferretti, J. J. (1987) In: Streptococcal Generics, Ferretti, J.J., and Curtis, R. III [Ed.] Proc. American Society for Microbiology.,Washy D.C. p. 143). It has been demonstrated that SK interacts with PGthrough a least two major loci, mapped between residues 16-51 and230-290 (Nihalani, D., Raghava, G. P. S., and Sahni, G., 1997, Prot.Sci. 6:1284), and probably also the region and around residues 331-332(Lin, F. L., Oeun, S., Houng, A., and Reed, G. L., 1996, Biochemistry35:16879); in addition the sequences at the C-terminal ends, especiallybefore the last 30-32 residues of the native sequence (Kim. J. C., Kim,J. S., Lee, S. H., and Byun, S. M., 1996, Biochem. Mol. Bio. Int.40:939. Lee, S. H., Jeong, S. T., Kim, I. C., and Byun, S. M., 997,Biochem. Mol. Bio. Int. 41:199. Fay, W. P., Bokka, L. V., 1998, Thromb.Haemost 79:.985) are important in generating the activator activityassociated with the complex. Since a primary consideration in designingthe SK-FBD chimeras was the engineering of a decreased, or kineticallyslowed, initial PG activation rate, we reasoned that either the C- orN-termini (or both, together) could be utilized to bear the FBDs in thehybrid structures, and that the presence of such ‘extra’ domains in SK,either full-length or already truncted to the most functionally relevantregions of human fibronectin that can independently bind fibrin underphysiological conditions (detailed earlier) and would also suitablyretard and/or delay the PG activation rates observed normally withnative SK and PG by interfering in the interactions of SK with PG togenerate a functional activator complex. If the polypeptide in betweenthese two distinct parts constituting the chimera were sufficientlyflexible, proteolytic scission in this region would then result in theremoval of the retarding/inhibiting portion (FBD component) from theSK-FBD hybrid and lead to a burst of PG activation after an initialdelay. This proteolysis could be mediated by the small amounts ofendogenous plasmin generated in the vicinity of the pathological clot byintrinsic plasminogen activator/s of the system, such as TPA, urokinaseetc already present in the circulatory system. Indeed, this premise wasborne out by experimentation, which showed that the lag times of PGactivation by the SK-FBD chimeras disclosed in this invention weredirectly governed by plasmin-mediated proteolysis of the hybrid proteinsleading to the liberation of the FBD portion from the SK-FBD followed byrapid PG activation by the SK. The direct implication of this functionalproperty in a plasminogen activator is that once injected into the body,the protein could then traverse in an inactive state through thecirculatory system and bind to the pathological clot by virtue of thefibrin affinity imparted by the fibrin binding domains thereby obviatingor minimizing sytemic PG activation. Thus, if the thrombolytic agenttraverses the circulation prior to this activation (which is known torequire 3-5 minutes in the human circulation), the fibrin affinity inthe chimera would allow it to bind to the clot, thereby helping tolocalize the PG activation in and around the immediate vicinity of thethrombus.

The amino acid sequence of human FN is known to be composed of threetypes of homologous repeats (termed type-1, type-2 and type-3), of whichthe FBDs at the amino terminus of FN are made of five type-1 repeats,each approximately 50 residues long and containing two disulfidebridges. The C-terminus of FN also has three type-2 homology repeatsthat are involved in fibrin-FN interactions. Therefore, altogether, alarge portion of the FN molecule, representing the several N- andC-terminally located FBDs, could be linked with SK if all of the fibrininteracting regions need to be incorporated into the contemplated SK-FBDchimeras. However, such a design produces a chimeric protein that is notonly too bulky, but also decreases the probability for the polypeptideto fold into a biologically active conformation due to the presence of alarge number of S—S bridges that may form non-native, intra- andinter-molecular dude bonds. Instead, a potentially more worthwhileproposition is to seek miniaturised but, nevertheless, functionallyactive combinations of selectively truncated regions of SK and/or FBDs.

OBJECTS OF THE INVENTION

The main objective of the invention is to provide a novel chimericstreptokinase-based plasminogen activator protein molecule withadvantageous characteristics of improved fibrin affinity and plasminogenactivation kinetics characterized by an initial lag of several minutes'duration (5 to 30 minutes) prior to attainment of full-blown capabilityof PG activation.

Another object is to provide a system for the production of the hybridpolypeptides, which includes DNA segments/polynucleotide blocks encodingthe polypeptides, plasmids containing these genetic elements capable oftheir expression into protein, as well as microorganisms transformedwith these plasmids.

Yet another object is to design a process for the production of thehybrid plasminogen activator protein molecules in pure and biologicallyactive form for clinical and research applications.

Another object is to provide an improved process for the intracellularproduction of large quantities of SK, or its modified forms, in E. coliusing an altered DNA polynucleotide, and obtain these in a pure andbiologically active form.

A further object is to provide pharmaceutical compositions comprisingnovel chimeric streptokinase-based plasminogen activators of theinvention and pharmaceutically acceptable carriers.

SUMMARY OF THE INVENTION

The invention provides hybrid plasminogen activator comprising apolypeptide bond union between streptokinase (SK), or modified forms ofSK, or suitable parts thereof, which are capable of plasminogen (PG)activation, with fibrin binding regions of human fibronectin selectedfrom the pair of fibrin binding domains 4 and 5, or domains 1 and 2 ormodified forms thereof, to achieve various motifs for joining the fibrinbinding domains with streptokinase or its modified forms, so that thehybrid plasminogen activator possesses the ability to bind with fibrinindependently and also characteristically retains a plasminogenactivation ability which becomes evident only after a pronouncedduration, or lag, after exposure of the plasminogen activator to asuitable animal or human plasminogen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a hybrid streptokinase-based plasminogenactivator prepared by conventional recombinant DNA techniques e.g.,those described in ‘Sambrook et al., Molecular Cloning: A LaboratoryManual’ (II^(nd) Ed., Cold Spring Harbor Press, 1989) and ‘DNA Cloning’(vol. I to III) (Glover, D. M., [Ed.], IRL Press Ltd., London, 1987),among several other manuals/compendia of protocols, and the techniquesof protein purification and characterization, in particular the variouschromatographic methods employed conventionally for purification anddownstream processing of natural and recombinant proteins and enzymesviz., hydrophobic interaction chromatography (HIC), ion-exchange and gelfiltration chromatographies, and affinity chromatographic techniqueswell-known in the field of protein biochemistry (e.g., in this regard,reference may be made to (i) Protein purification. Principles, highresolution methods and applications. Janson, J -C., and Ryden, L.,[Ed.], VCH Publishers Inc., New York, 1989; (ii) Process Chromatography:A practical guide. Sofer, G. K., and Nystrom, L. E., [Ed], AcademicPress, New York, 1989).

The advantage of the present invention lies in its disclosure of thedesign of structurally defined hybrid DNA polynucleotide constructs inwhich the translational in-frame fusion of the DNAs encoding SK or itsmodified forms, and the minimally essential parts of thefibronectin-encoding DNA polynucleotides essential for significantfibrin affinity on their own, such as those FBDs that possessindependent fibrin binding capability (such as finger domains 4 and 5 ofhuman fibronectin) is carried out in such a configuration that confersthe additional property of a time-delayed plasminogen activation in theresultant hybrid protein molecules. The latter are expressed bytranslation of the hybrid polynucleotides formed between the SK-encodingDNA and the FBD encoding DNA in a suitable host cell such as a bacteriumyeast, animal, plant cell etc. The resultant hybrid proteins, containingSK and FBD portions fused to each other through polypeptide linkages,can be isolated in pure form by conventional methods of proteinpurification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of different chimeric proteins preparedby the fusion of SK and FBDs.

FIG. 2. Map of plasmid pJKD-8, containing SK gene from S. equisimilisH46A.

FIGS. 3.1 to 3.2 shows the DNA and protein sequence of streptokinase ofS. equisimilis H46A (SEQ ID NOs: 1 and 2, respectively)(GeneBankaccession number: gb/K02986/STRSKC).

FIG. 4. Partial restriction enzyme map of DNA encoding for SK.

FIG. 5. Map of plasmid pFH-6, containing FBD 1 to 5 encoding sequencesaccording to Kornhblitt, A. R., Umerzawa, K., Vibe-Pedersen, K. andBaralle, F. E., (1985) EMBO J. 4:1755.

FIG. 6. DNA and protein sequence of the gene-segment encoding for FBDs1-5 of human fibronectin (SEQ ID Nos.: 3 and 4, respectively) (the DNAsequence has been obtained from EMBL; the file and accession no.'s areID-HSFIBI and X 02761, K 00799, K 02273, X 00307, X 00739).

FIG. 7. Restriction enzyme map of DNA encoding the five N-terminallylocated FBDs of human fibronectin.

FIG. 8. Map of plasmid pET23(d).

FIG. 9. Flow chart schematically depicting the main steps in theconstruction of a plasmid vector for the expression of the native SKgene of S. equisimilis H446A.

FIG. 10. Flow diagram schematically depicting the main steps involved inthe repair of the vector pET23(d)SK i.e construction of expressionvector pET23(d)SK-NTRN (NTRN: abbrev. form for N-terminally repairedwith native sequence).

FIG. 11. Nucleotide sequence of SK-NTRN gene (SEQ ID NO:5).

FIG. 12. Predicted secondary structure of native (A) and translationallysilently modified (B) 5′-ends of the SK gene sequence (SEQ ID NO.:6).

FIG. 13. Schematic flow diagram depicting the main steps in theconstruction of a plasmid vector [pET23(d)SK-NTR] for the intracellularhyper-expression of a semi-synthetic SK gene in which the 5′-end of theopen-reading-frame for SK was selectively modified in translationallysilent manner at the DNA level, so that it encoded for the primarystructure of S. equisimilis SK.

FIG. 14. Nucleotide sequence of SK-NTR gene.

FIG. 15. Schematic depiction of the intergenic region of the chimericSK-FBD(4,5) gene (above: SEQ ID NO:8; below: SEQ ID NO:7) highlightingthe design of a gly-gly-gly sequence, a transglutaminase cross-linkingsite and several unique restriction enzyme sites wherein differentinter-genic cassettes can be conveniently swapped into this region. Alsoshown is the location of the natural Bsm I site in the SK gene which wasexploited as the common junction point for joining the FBD sequences tothe SK gene.

FIG. 16. Flow diagram depicting the main steps in the construction ofplasmid pSKMG400, containing the SK-FBD(4,5) hybrid DNA block composedof FBD(4,5) sequences linked to the intergenic sequences at its 5′-end,and the SK gene fused in-frame at the 3′ end.

FIG. 17 a. Scheme depicting the cloning of the hybrid SK-FBD(4,5)cassette into, pET23(d)SK-NTR for intracellular expression ofSK-FBD(4,5) chimeric protein in E. coli.

FIG. 17 b. DNA sequencing data of SK-FBD(4,15) hybrid cassette in T7expression vector, pET23(d) (SEQ ID NO:9).

FIG. 18. Schematic flow diagram for cloning of SK-FBD(1,2) hybrid genein pBluescript to obtain pSKMG400-FBD(1,2).

FIG. 19 a. Schematic description of steps involved in the cloning of thehybrid gene-construct SK-FBD(1,2) into expression vector pET23(d)SK-NTRfor intracellular expression of SK-FBD(1,2) chimera in E. coli.

FIG. 19 b. DNA sequencing data of SK-FBD(1,2) hybrid cassette in T7expression vector (SEQ ID NO: 10).

FIG. 20. Scheme of steps involved in the construction of hybrid geneblock composed of DNA encoding for FBD(4,5) and residues 1-63 of SK bythe Overlap Extension PCR technique.

FIG. 21 a. Scheme depicting steps involved in the cloning of theFBD(4-5)-SK gene block for expression of FBD(4,5)-SK chimera in E. coli.

FIG. 21 b. DNA sequencing data of FBD(4,5)-SK gene block as present inthe T7 expression vector pET23(d)-FBD(4,5)-SK (SEQ ID NO:11).

FIG. 22 a. Flow chart depicting schematically the steps involved in theconstruction of FBD(4,5)-SK-FBD(4,5) hybrid gene in pET23(d) expressionvector.

FIG. 22 b. DNA sequencing data of FBD(4,5)-SK-FBD(4,5) gene block aspresent in the T7 expression vector pET23 (d)FBD(4,5)-SK-FBD(4,5) (SEQID NO:12).

FIG. 23. Purification of SK-FBD(4,5) protein expressed in E. coli by aone-step affinity chromatographic procedure.

FIG. 24. Clot lysis by purified SK-FBD(4,5) chimeric protein in a plasmamilieu. FIG. 24 shows results with: closed triangles, 100 nm SK; opentriangles, 50 nm SK; closed circles, 200 nm SK-FBD(4,5); closed squares,100 nm SK-FBD(4,5); open squares, 50 nm SK-FBD(4,5).

Accordingly, the present invention provides a hybrid plasminogenactivator comprising a polypeptide bond union between streptokinase(SK), or modified forms of SK, or suitable parts thereof, which arecapable of plasminogen (PG) activation, with fibrin binding regions ofhuman fibronectin selected from the pair of fibrin binding domains 4 and5, or domains 1 and 2, or modified forms thereof, so that the hybridplasminogen activator possesses the ability to bind with fibrinindependently and also characteristically retains plasminogen activationability which becomes evident only after a pronounced duration, or lag,after exposure of the plasminogen activator to a stable animal or humanplasminogen. FIG. 1 describes the different designs of the SK-FBD hybridproteins schematically as disclosed in the invention.

In an embodiment, the invention provides a hybrid plasminogen activatorwhich carries out plasminogen activation only after a lag period varyingbetween 5 and 30 minutes after exposure of the plasminogen activator toa suitable animal or human plasminogen.

In a further embodiment, the invention provides a DNA segment encodingthe hybrid plasminogen activator.

In yet another embodiment, the invention provides an expression vectorcontaining a DNA segment encoding the hybrid plasminogen activator.

In another embodiment, the invention provides prokaryotic or eukaryoticcells, transformed or transfected with expression vectors, and capableof expressing the hybrid plasminogen activators.

The invention further provides a method for the preparation of hybridplasminogen activators possessing useful plasminogen activationcharacteristics, said method comprising the steps of:

(a) Preparing a first DNA encoding a nucleotide sequence forstreptokinase or any of its modified forms, by conventional biochemicalor chemical methods or appropriate combinations thereof, to produce atranslational product which is a polypeptide that can activateplasminogen.

(b) Preparing a second DNA polynucleotide by known biochemical orchemical methods or appropriate combinations thereof, that encodes forthe fibrin binding domains selected from the pair of fibrin bindingdomains 4 and 5, or domains 1 and 2, or their modified forms, that arecapable of conferring affinity and/or specificity for fibrin, andlinking these to another DNA molecule that is capable of undergoingmultiplication in a suitable host cell,

(c) Construction of hybrid polynucleotides, between the first DNAencoding for streptokinase, or its modified forms, that encode for apolypeptide capable of plasminogen activation, with the second DNAencoding for the fibrin binding domains (FBD) of fibronectin byconventional methods, in the native translational codon frame startingwith an initiator codon, and joining of the hybrid polynucleotide into arecipient DNA molecule, such as a plasmid capable of autonomousreplication in a host cell, or capable of integrating into the genomicDNA of a suitable host cell, and expressing the hybrid protein therein,

(d) Introducing the DNA containing the hybrid polynucleotide constructsobtained in step (c) into an appropriate host, selected from the groupcomprising E. coli, Bacillus sp., yeast, fungus, plant, animal cell byconventional methods,

(e) Culturing the host cells expressing the SK-FBD chimericpolynucleotide using known procedures,

(f) Isolating components of the culture, selected from, extracellularfluid from fermentation, intracellular milieu of the host cell orcombinations thereof, that harbour the expressed chimeric polypeptide inan enriched from and then partially purifying the chimeric polypeptidesusing conventional procedures selected from the group comprisingcentrifugation, ultrafiltration, precipitation with salts or organicsolvents etc, or chromatography on suitable media, or combinationsthereof,

(g) Refolding the hybrid polypeptide to a biologically active andstructurally intact form, if required,

(h) Further purifying the biologically active hybrid polypeptide fromthe relatively crude or partially pure material/s or cell-free lysateobtained at step (f), or (g) above, after refolding, using conventionalmethods of protein purification, or by affinity chromatography on asuitable matrix comprising immobilized fibrin or fibrinogen, or specificantibodies that recognize and bind with the active, biologically activehybrid proteins,

In an embodiment, the invention provides a method for the production ofthe hybrid plasminogen activator proteins including DNAsegments/polynucleotide blocks encoding the polypeptides, plasmidscontaining these genetic elements capable of their expression intoprotein, as well as microorganisms or other suitable host cellstransformed with these plasmids.

In another embodiment, the invention provides a method for theproduction of the hybrid plasminogen activator molecules in pure andbiologically active form for clinical and research applications.

In another embodiment, the invention provides a method for theintracellular production of large quantities of SK, or its modifiedforms in bacteria such as E. coli, using, a polynucleotide block that isaltered as compared to that of the natural DNA sequence encoding for SKor its modified forms, and obtain these in a pure and biologicallyactive form.

In yet another embodiment, the invention provides a method wherein the5′-end of the SK-encoding polynucleotide utilized for expression of SK,or its modified forms such as the SR-FBD chimeric polypeptides, ismodified, as exemplified by the DNA sequence provided in FIG. 13, bymutagenesis by known biochemical or chemical DNA synthesis techniques,or their combination, such that the secondary structure-formingcapability (e.g., the intramolecular hydrogen bonding capability) of itstranscript is diminished, resulting in increased efficiencies ofexpression of SK or its modified forms such as SK-FBD chimeras in theheterologous host cell.

In another embodiment, the invention provides a method wherein the5′-end of the SK-encoding DNA or its modified forms such as the SK-FBDchimeric polypeptides is modified by mutagenesis by known biochemical orchemical DNA synthesis techniques, or a suitable combination thereof, insuch manner that the codons utilized in the DNA polynucleotide artcompatible with those frequently utilized in E. coli or the host cellused for the expression of the genes.

In another embodiment, the invention provides a method wherein the DNAencoding those fibrin binding domains that possess independent fibrinbinding capability are fused in the correct translational frame at the5′-end of the SK-encoding DNA, after a translational start codon, andthen expressed into protein of the form exemplified in FIG. 1C, refoldedoxidatively and isolated in the purified form, to obtain the desiredcharacteristic in the chimera viz, characteristic PG activationproperties characterized by an initial lag in the PG activation ratestogether with significant fibrin affinity.

In another embodiment, the invention provides a method wherein thefibrin binding domains are fused in-frame at the C-terminal end of theSK, or its modified form, to obtain a hybrid SK-fibrin binding domainprotein that contains selected fibrin binding domains at the C-terminalend of the SK portion of the chimera after expression of the hybrid DNAin a suitable host cell, as exemplified in FIGS. 1A and 1B, to obtainthe desired characteristic in the chimera viz., characteristic PGactivation properties characterized by an initial lag in the PGactivation rates together with significant fibrin affinity.

In another embodiment, the invention provides a method wherein thefibrin binding domains are fused through polypeptide linkage at theC-terminal end of the SK devoid of upto 45 amino acids, preferably 31amino acid residues. Thus, a hybrid SK-FBD protein is generated thatcontains selected fibrin binding domains fused at the C-terminal end ofa truncated SK, thus yielding a chimeric protein that has both fibrinaffinity as well as delayed PG activation properties.

In yet another embodiment, the invention provides a method whereinfibrin binding domains are fused at both the ends of SK, or its modifiedforms that retain a plasminogen activator ability, simultaneously (inthe configuration represented as ‘TFD-SK-FBD, as schematically depictedin FIG. 1, D) to achieve the desired functionality in the hybridconstruct viz, characteristic plasminogen activation propertiescharacterized by an initial lag in the PG activation rates together withsignificant fibrin affinity.

In another embodiment, the invention provides a method wherein thenovel, chimeric polypeptides are expressed in E. coli or other suitablehost cells.

In another embodiment, the invention provides a method wherein SK or itstruncated form/s are fused through polypeptide linkages with the fibrinbinding domains known to possess independent fibrin binding capabilitythrough a short linker peptide region comprising of a stretch of aminoacid sequence that is not conformationally rigid but is flexible, suchas those predominantly composed of Gly, Ser, Asn, Gln and similar aminoacids.

In another embodiment, the invention provides a method wherein SK or itsmodified forms are fused with fibrin binding domains through a “linker”peptide composed of amino acid sequences that provide varying levels oflocal conformational flexibility by incorporating sequences that foldinto relatively rigid secondary structures such as beta-turns so as toobtain different chimeric PG activator proteins with desirable initiallag-times in their plasminogen activation kinetics.

In another preferred embodiment, the SK-FBD hybrid polypeptides areexpressed in E. coli using known plasmids under the control of strongpromoters, such as iac, trc, trp, T7 RNA polymerase and the like, whichalso contain other well known features necessary to effect high levelexpression of the incorporated DNA polynucleotides encoding for thehybrid Streptokinase-fibrin binding domain polypeptides e.g.Shine-Delgarno sequence, transcription terminating signals etc.

In yet another embodiment, SK or its truncated forms are fused either atthe amino or C-termini, or both, through polypeptide linkages with theFBDs known to possess independent fibrin binding capability, such asdomains 4 and 5, through short linker regions, as described above, thatcontain amino acid sequence/s providing varying levels of localconformational flexibility to the linker segment between the SK and FBDportions of the hybrid protein/s.

In yet another embodiment, various chemical or physical agents, such asiso-propyl-beta-D-thio galacto pyranoside (IPTG), lactose, low or hightemperature change, change in salt or pH of medium, ethanol, methanol,and the like, are used to induce high levels of the SK or the varioushybrid polypeptides in the host cell in which the hybrid polynucleotidesare being expressed.

In another preferred embodiment, the hybrid SK-FBD polynucleotides areexpressed in E. coli.

In yet another embodiment, the E. coli cells are lysed by chemicaltreatment such as the use of chaotropic salts e.g. guanidiniumhydrochloride and the like, to effect the liberation of the SK or itsmodified hybrid forms, which are then purified using conventionalprocedures.

In yet another embodiment, the invention provides a method wherein thehost E. coli cells are lysed by chemical treatment such as chaotropicsalts e.g., guanidinium hydrochloride and the like, to effect theliberation of the SK or its modified chimeric forms, followed bypurification using conventional methods.

In another embodiment, the invention provides a method where the crudecell-lysates obtained, using either conventional methods or by employingchaotropic salts, from cells elaborating the chimeric polypeptides aresubjected to air oxidation to refold chimeric polypeptides to theirbiologically active conformations containing the native cystineconnectivities.

In another embodiment, the invention provides a method wherein the crudecell-lysates obtained using either conventional methods selected fromthe group consisting of enzymatic lysis of cells, ultrasonic lysis,lysis by mechanical means or by employing chaotropic salts, from cellselaborating the chimeric polypeptides are subjected to oxidation andrefolding using a mixture of reduced and oxidized glutathione of asuitable redox potential that allows the chimeric polypeptides to refoldto their biologically active conformations.

In yet another embodiment, the invention provides a method wherein therefolding reaction is carried out in the presence of immobilized fibrinto promote a more efficient ligand-induced refolding of the epitopesresponsible for fibrin affinity in the said chimeric polypeptides, andconsequently higher yields of the biologically active chimeric proteinconstructs.

In another embodiment, the invention provides a method wherein thebiologically active chimeric polypeptides are purified selectively fromother proteins, or unfolded SK-FBD polypeptides, by affinitychromatography on immobilized fibrin(ogen) e.g. fibrin- orfibrinogen-agarose.

In another embodiment, the invention provides a method wherein achimeric plasminogen activator protein is used as a medicant for thetreatment or propylaxis of thrombolytic diseases. The activator may beformulated in accordance with routine procedures as pharmaceuticalcomposition adapted for intravenous administration to human beings, andmay contain stabilizers such as human serum albumin, mannitol ect,solubilizing agents, or anesthetic agents such as lignocaine and thelike.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising a hybrid plasminogen activator and stabilizerssuch as human serum albumin, mannitol etc, solubizing agents, anestheticagents.

In a further embodiment, the pharmaceutical composition of the inventioncomprises a chimeric plasminogen activator protein used as a medicantfor the treatment or prophylaxis of thrombolytic diseases andpharmaceutically acceptable carriers. The activator may be formulated inaccordance with routine procedures as pharmaceutical composition adaptedfor intravenous administration to human beings, and may containstabilizers such as human serum albumin, mannitol etc, solubalizingagents, or anesthetic agents such as lignocaine and the like.

The DNA polynucleotides encoding the various streptokinase-fibrinbinding domain hybrid constructs depicted schematically in FIG. 1 can bemade utilising rDNA and selective DNA amplification techniques (e.g.,the well-known polymerase chain reaction technique, abbreviated PCR)(reference, in this regard may be made to Saiki, R. K., Scharf, S.,Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Amheim, N.,1985, Science 230: 1350; Mullis, K. B., and Faloona, F., 1987, Methodsin Enzymol. 155.335). The hybrid genes are then expressed inheterologous hosts such as bacteria (e.g. E. coli), or other suitableorganisms, to obtain the chimeric polypeptides. Bacterial host cells (E.coli XL Blue) harbouring the various plasmid constructs expressing thedifferent SK-FBD hybrid proteins (see Examples section) have beendeposited in the Microbial Type Culture Collection (MTCC), Institute ofMicrobial Technology, Chandigarh (a constituent laboratory of C.S.I.R.,India). The accession numbers of these constructs are: BPL 0013 for thepET 23(d)-NTR-SK-FBD(4,5) construct (see FIG. 17 a for map of thisplasmid); BPL 0014 for pET 23(d)SK-NTR-FBD(1,2) (see FIG. 19 a); BPL0015 for pET23(d)FBD(4,5)-SK (see FIG. 21 a for map of this plasmidconstruct); BPL 0016 for pER23(d)FBD(4,5)SK-FBD(4,5) (See FIG. 22 a formap); 0017 for pET23(d)SK-NTR (see FIG. 13 for map of this plasmidconstruct). The proteins expressed from these plasmids can be expressedin suitable host cells (e.g. E. coli BL 21) and then purified to renderthem substantially free of other components derived from the hostproducer cells. In case the polypeptide is expressed in a host systemnot capable of efficient re-oxidative folding of the primarytranslational product/s of the hybrid genes e.g. E. coli, anintermediate in vitro refolding step is introduced subsequent to theexpression step. Alternatively, the hybrid constructs can be expressedin cell systems capable of efficient oxidative refolding oftranslational products e.g. yeast, animal cells etc.

The DNA polynucleotide encoding for SK was first cloned in a bacterialplasmid in E. coli after isolation from the wild-type S. equisimilisgenomic DNA according to known procedures (Malke, H., and Ferretti, J.J., 1984; Proc. Nat'l Acad. Sci. 81: 3557) and in recent researchpublications available in the public domain (Pratap, J., Kaur, J.,Rajamohan, G., Singh, D., and Dikshit, K. L., 1996, Biochem. Biophys.Res. Commun. 227:303). In the process of the present invention, the DNAcorresponding to the translational open-reading-frame (ORF) of SK hasbeen further modified with respect to its 5′-coding sequences so thatafter cloning in an expression vector under the control of a strongpromoter, large quantities of biologically active SK are producedintracellularly. The DNA sequence of the SK gene from Streptococcusspecies (American Type Culture Collection accession No. 12449; thisstrain has served as the producer strain for numerous studies onstreptokinase, and is often referred to as Streptococcus equisimilisH46A in the scientific literature). The corresponding amino acidsequence of the mature protein are provided in FIG. 3. The restrictionenzyme map of the SK-encoding DNA is provided in FIG. 4.

The DNA polynucleotide sequences encoding for the fibrin binding domainsof human fibronectin were selectively amplified from known plasmidscontaining cloned cDNA for the FN gene. Kornhblitt, A. R.,Vibe-Pedersen, K., and Baralle, F. E., 1983. Proc. Natl. Acad. Sci.80:32 18 have cloned the cDNA encoding for the human fibronectin (FN)gene in a plasmid vector in E. coli (pFH1). This cDNA extends approx.2.1 kb from the poly-A tail of the mRNA of fibronectin, aroundone-fourth of the estimated size of the human FN message (approx. 7900nucleotides). By further mRNA “walking” type of experiments, theseinvestigators carried out the coytnuction of longer cDNA clones usingsynthetic oligonucleotides complementary the DNA of clone pFH1. By thismethod, cDNAs corresponding to the complete FN mRNA were prepared andcloned in several vectors (Kornhblitt A. R., Umezawa, K, Vibe-Pedersen,K., and Baralle, F. E., (1985) EMBO J. 4:1755). One such plasmid (pFH6)contained the entire sequences coding for the FBDs of the N-terminalregion of human FN (as represented in FIG. 5 showing the map of thisplasmid, and in FIG. 6 showing the nucleotide and amino acid sequence ofthe FBD regions contained in this plasmid and FIG. 7 for its restrictionenzyme map). Plasmid pFH6 served as the source for these sequences inthe construction of the SK-FBD hybrids. The fibrin binding domainslocated in the N-terminal region of human FN gene were selectivelyamplified by PCR using specially designed oligonucleotide primers thathybridized with DNA sequences flanking the FBD DNA segments to beamplified. These primes also contained non-hybridizing sequences attheir 5′-ends that provided the intergenic sequence (i.e. between the SKand FBD DNA) as well as a restriction site through which the amplifiedDNA could be ligated with the SK gene in-frame in a plasmid vector. Thecloned hybrid gene was then expressed in E. coli so as to produce largequantities of the chimeric polypeptide. This protein was then isolatedfrom the E. coli cells and subjected to a process of purification andrefolding to a biologically active form. Similarly, different designs ofthe SK and FBD hybrids were then constructed using recombinant DNAmethods, expressed, and isolated in biologically active, purified forms.Analysis of the properties of these proteins established that theseindeed possessed the functional properties expected from their designi.e. plasminogen activation ability as well as fibrin selectivity. Theyalso displayed the additional desired property of a very slow initialkinetics of PG activation, which, after a lag varying between 5-30minutes, depending on the construct, was overcome, leading to high ratesof PG activation comparable to native SK.

The invention and its embodiments are illustrated by the followingexamples, which should not be deemed to limit the scope of the inventionin any manner. Various modifications that may be apparent to thoseskilled in the art are deemed to fall within the scope of the invention.

I. General Methods Used in Examples

1. Recombinant DNA methods: In general, the methods and techniques ofrDNA well known in the area of molecular biology were utilised. Theseare readily available from several standard texts and protocol manualspertaining to this field of the art, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual 2^(nd) edition, Cold SpringHarbor Press, New York., 1989; McPherson, M. J., Quirke, P., and Taylor,G. R., [Ed,] PCR: A Practical Approach. IRL Press, Oxford., 1991).However, pertinent details in the context of specific experimentsdescribing the present invention, particularly where modifications wereintroduced to established procedures, are indicated in the Exampleswherever relevant.

Casein-plasminogen overlay for detection of SK activity: bacterialcolonies producing streptokinase can be routinely detected by overlay ofcasein and human plasminogen in soft agar (Malke, H., Ferretti, J. J.,1984, Proc. Natl. Aca. Sci. 81:3557). Ten ml soft agarose mixtureconsisting of 0.8% agarose, 10% skimmed milk, approx. 200 ug of humanplasminogen, 150 mM NaCl, and 50 mM Tris-Cl (pH 8.0) is poured on top ofthe plates. The plates are incubated at 37° C. for 2-6 h. Positivestreptokinase activity is indicated by the appearance of zones ofclearance around the colonies (halo formation).

Zymography: proteins from total cell lysates are separated on 10%SDS-PAGE. After completion of electrophoresis rum, gel is washed with2.5% Triton X-100 to remove any SDS. It is then thoroughly rinsed withstandard buffer 0.05 M Tris Cl (pH 7.5) for Triton X-100 removal. Thegel is laid on 0.6% agarose plate containing 10% skimmed milk and 0.5mg/ml human plasminogen. After incubating at 37° C. for 2-3 h, an activeSK band is visualized as a clear band.

4. SDS-PAGE analysis of proteins: SDS-PAGE is carried out, essentiallyaccording to Laemmli, U.K., 1970, Nature 227:680 with minormodifications, as needed. Briefly, protein samples are prepared bymixing with an equal volume of the 2× sample buffer (0.1 M Tris Cl, pH6.8; 6% SDS; 30% glycerol; 15% bcta-merecaptoethanol and 0.01%Bromophenol Blue dye). Prior to loading onto the gel, the samples areheated in a boiling water bath for 5 min. The discontinuous gel systemusually has 5% (acrylamide conc.) in the stacking and 10% in theresolving gel. Electrophoresis is carried out using Laemmli buffer at aconstant current of 15 mA first, till the samples enter the gel and then30 mA till the completion. On completion of electrophoresis, gel isimmersed in 0.1% coomasie Blue R250 in methanol:acetic acid:water(4:1:5) with gentle shaking and is then destained in destaining solution(20% methanol and 10% glacial acetic acid) till the background becomesclear.

5. Immuno-assay of Western blotted-proteins: Wester blotting of theproteins from E. coli carrying plasmid encoded intracellularstreptokinase is carried as detailed (Towbin, H., Stachelin T., Gordon,T., 1979, Proc. Natl. Acad. Sci. 76: 4350). The cultures are grown to600 nm of 0.5-0.6, and are induced with 1-5 mM IPTG. The cells arecentrifuged. The pellet is resuspended in cell lysis buffer and thesupernatant obtained after high-speed centrifugation. These fractionsare resolved on the 10% SDS-PAGE. The gel is equilibrated with thetransfer call buffer (25 mM Tris, 175 mM glycine in 20% methanol) and isblotted electrophoretically in to the nitrocellulose membrane at 50 Vfor 3 h. The blot is blocked with 5% skimmed milk (Difco) in PBS(Phosphate buffer saline) for 14-16 h at 4° C. The blot is furtherwashed with 0.1% Tween-20 in PBS. The blot is incubated with the anti-SKantibodies (raised against pure S. equisimilis SK in rabbit) in 40 ml ofPBS containing 5% skimmed milk for 3 h at room temperature with gentleshaking. The blot is washed with 0.1% Tween 20 in PBS three times for 15minutes each. Again it is blocked with PBS-skimmed milk for 15 min withgentle shaking at room temperature and further incubated withperoxidase-conjugated goat anti-rabbit immunoglobulins at a dilution of1:5000 in 20 ml of PBS-skimmed milk (5%) for two 2 h at room temperaturewith gentle shaking the filter is again washed with 0.1% Tween 20 in PBSfor three times (15 min each). The colour reaction for HRP-linkedsecondary antibodies is carried out by immersing the blot in 10 ml ofreaction buffer solution having 10 mg of DAB (di-amino benzidine) andimidazole each. The reaction is terminated by washing with distilledwater.

6. Streptokinase assay using chromogenic peptide substrate: plasminogenactivator activity of streptokinase is assayed according to Jackson, K.W., Esmon, N., Tang, T., 1981, Methods in Enzymology 80: 387. Onehundred ul of appropriately diluted streptokinase samples, 25 ul ofsample buffer (0.15 M Tris-Cl buffer, pH 7.5) and 15 ul of humanplasminogen solution (0.5 mg/ml in 0.05 M Tris-Cl, pH 7.5) are mixedtogether. The tubes are incubated at 37° C. for 15 min, after which 18ul of NaCl (1.77M in 0.032 M Tris-Cl, pH 7.5) is added. The amount ofplasmin generated in the first stage is measured by further addition of12 ul of plasmin-specific chromogenic substrate, Chromozyme-PL(Boehringer-Mannheim, Germany), 5 mg/ml in water, and the tubes areagain incubated at 37° C. for 10 min. After this incubation, 0.4 mlacetic acid (0.2 M) is added to terminate the reaction. The release ofyellow-colored 4-nitroaniline is monitored at 405 nmspectrophotometrically. Appropriate dilutions of S. equisimilisstreptokinase obtained from WHO, Hertfordshire, U.K. is used as areference standard for calibration of international units in the unknownpreparation. Protein concentration is estimated according to the methodof Bradford., M. M., 1976, Anal Biochem. 72: 248) using BSA as astandard. Bradford's reagent (Biorad Inc., USA) is utilized according tothe manufacturer's instructions. For estimating the concentration,protein-samples in phosphate buffer are made to 800 ul. To this, 200 ulBradford's reagent is added and is mixed thoroughly. The reaction ismaintained at room temperature for 5 min and absorbance at 595 nm ismonitored. The specific activity for PG activation (I.U./mg protein) ofan unknown preparation of SK or SK-FBD is thus determined from the SKassay and protein estimation data.

7. Fibrin clot assay for SK: This test is performed to determine theclot lysis ability of any thrombolytic drug, such as streptokinase,urokinase or tissue plasminogen activator, and is adapted from BritishPharmacopia (1980 edition).

Reagents: (i) 100 mM citrate phosphate buffer. pH 7.1 containing 0.8%BSA (referred to as buffer-1). (ii) Bovine fibrinogen (Cohn Fraction-I,obtained from Sigma Chemical Co., St. Louis, USA), 2.5 mg/ml prepared inbuffer-1). (iii) Bovine thrombin (obtained from Sigma as a lyophilizedpowder). Stock solution of SK, 500 I.U./ml, prepared in sterile waterand stored in aliquots of 50 ul each at −70° C. Before use, one aliquotis thawed and diluted to 50 I.U./ml in buffer-1. (iv) Human plasminogen(Boehringer Mannheim, Germany) stock 1 mg/ml, prepared in sterile water.Stored in aliquots of 100 ul each at −70° C.; (v) Standard SK (fromW.H.O., obtained from Dr. P. J. Gaffney, Division of Haematology,N.I.B.S.C., Blanche Lane, Potters Bar, Hertfordshire, EN 6 3QG, U.K.).The standard SK vial is composed of 700 international units of SK (inlyophilized form alongwith stabilizers). A complete vial should bedissolved in 700 ul of sterile dist. water to obtain a concentration of1000 I.U./ml. The dissolution should be carried out either at 4° C. orby keeping all the solutions on ice. The dissolved SK is then aliquotedinto convenient sizes and stored at −70° C. Prior to carrying out clotlysis assay an aliquot of 1000 I.U/ml (stock) is thawed and dilutedfurther in cold buffer-1 on ice. Dilutions (A to D, below) are preparedserially in the following way using a new pipette tip for each transfer.A. 10 ul of stock+990 ul of buffer−1=10 I.U./ml.B. 500 ul of A+500 ul of buffer−1=5 I.U./ml.C. 500 ul of B+500 ul of buffer−1=2.5 I.U./ml.D. 500 ul of C+500 ul of buffer−1=1.25 I.U./ml.(All dilutions are tempered at 37° C. prior to use in the clot test asare the other solutions to be used.)Two hundred ul of each dilution is used in the clot lysis reactionmixture. One unit of SK. (present in 200 ul of SK dilution B) is justsufficient to lyse a standard fibrin clot in approximately 5 min at 37°C.Clot lysis test protocol: (a) Preparation of dot (negative control):During each step, the contents of the tube are gently mixed.Step 1: add 450 ul of buffer-1 to a small glass tube (0.8 mm internaldiameter).Step 2: add 50 ul of bovine thrombin (50 I.U./ml) solution to the tube.Step 3: add 100 ul of 1 mg/ml plasminogen to the solution in tube.Step 4: add 400 ul of 2.5 mg/ml fibrinogen to the solution in tube.Immediately after step 4, the tube is kept at 37° C. in a water bathwithout shaking. A standard clot forms within 30-40 seconds. The I.U./mlin the unknown is determined in a similar manner after appropriatedilution.B) Clot lysis with thrombolytic agent (SK): When clot lysis is to bepiled using standard SK, all the steps i.e. 1, 2 and 3 are carried outas described above, except that at step 1, only 250 uL of buffer-1 isadded. Also, at step 4, 200 ul from the appropriate dilution of SKcontaining 1-2 units (as described under Reagents, above) is premixedwith 400 ul of fibrinogen solution in a separate eppendorf tube, andrapidly equilibrated to 37° C. in water bath. This mixture is then addedto the clotting reaction at step 4, described above. The tube is thenincubated as previously. A clot is formed in the same or lesser time asabove, but is now followed by its lysis. The time for complete lysis isnoted down using a stop watch. The time for lysis depends upon theamount of SK used in the mixture. Lysis time by a particular unit ofstandard SK (i.e. lysis time of 5 min by 1 I.U. of SK) is taken as astandard. The unknown preparation of SK should be diluted appropriatelyto obtain a lysis time of approximately 5 min, which can then be used tocalculate the units of SK present in that unknown preparation.

8. Kinetic assays for determining the HPG activation by SK or SK-FBDchimeras: A one-stage assay method (Shi, G. Y., Cha, B. I., Chen. S. M.,Wu. D. H. and Wu, H. L., 1994, Biochem. J. 304:235. Wu, H. L., Shi. G.Y., and Bender, M. L., 1987, Proc. Natl. Acad. Sci. 84: 8292. Wohl. R.C., Summaria, L., and Robbins, K. C., 1980, J. Biol. Chem. 255:2005) wasused to measure the activation of HPG by SK or SK-FBDs. Varyingconcentrations of either SK or SK-FBD chimeric protein (10 nM-200 nM)were added to 100 ul-volume micro-cuvette containing 1 uM of HPG inassay buffer (50 mM Tris-Cl buffer; pH 7.5, containing 0.5 mMchromogenic substrate and 0.1 M NaCl). The protein aliquots were addedafter addition of all other components into the cuvette and bringing thespectrophotometric absorbance to zero. The change in absorbance at 405nm was then measured as a function of time in a Shimadzu UV-160 modelspectrophotometer.

9. Assay for determining the steady-state kinetic constants for HPGactivator activity of SK and SK-FBD constructs: To determine the kineticparameters for HPG activation, fixed amounts of SK or SK-FBD(4-5), 1 nM,were added to the assay buffer containing various concentrations of HPG(ranging from 0.035 to 2.0 uM) in the 100 uL assay cuvette as describedabove. The change in absorbance was then measured spectrophotometricallyat 405 nm for a period of 30-40 min at 22° C. The kinetic parameters forHPG activation were then calculated from inverse, Michalis-Menton,plots-by standard methods (Wohl, R. C., Summaria, L., and Robbins, K.C., 1980, J. Biol. Chem. 255:2005).

10. Radioactive fibrin clot preparation: 50 uL of (2.5 mg/ml) coldfibrinogen was mixed with 50 ul (9×10⁵ cpm) of ¹²⁵I-labelled fibrinogen(specific activity 8×10⁵ cpm/ug of fibrinogen) and added to the mixtureof 1.1 uM HPG and 0.025 units of human/bovine thrombin in 0.1 M citratephosphate buffer, pH 7.5 containing 0.8 percent BSA in a total volume of1 ml in a glass tube (1.3×12 cm). The clot was formed by incubating themixture at 37° C. for 2 min. The clot was then washed thrice with 2 mlcf TNT buffer (50 mM Tris-Cl buffer, pH 7.5, containing 38 mM NaCl and0.01 percent Tween-80) for 2 min at 37° C. As required thenon-radioactive fibrin clots were prepared by omitting ¹²⁵I-labelledfibrinogen from the clotting mixture.

11. Clot lysis in the presence of human plasma: ¹²⁵I-fibrin clot lysiswas carried out in the presence of 2 ml citrated human plasma containingdifferent concentrations of either SK or SK-FN (ranging from 100 to 200nM) at 37° C. The reaction tubes were rotated slowly at 37° C. and 0.1ml aliquots were removed at regular intervals to measure the soluble¹²⁵I-fibrin degradation products by measuring the amount ofradioactivity released using a gamma counter. The total radioactivity ofeach clot was determined by measuring the radioactivity of therespective tube before taking out any aliquots.

12. Clot lysis in the presence of human fibrinogen: ¹²⁵I-fibrin clotlysis was also carried out in the presence of various concentrations ofhuman fibrinogen (ranging from 1 to 4 mg/ml) containing 100 nM of eitherSK or SK-FN. Clot lysis was also performed in the presence of fixedfibrinogen concentration (2 mg/ml) and different concentrations of SK orSK-FBD protein construct (ranging from 50 to 200 nM). The reactions wereincubated at 37° C. with gentle shaking and the release of ¹²⁵I-fibrindegradation products as a function of time was measured as describedpreviously.

EXAMPLES Example 1

High level intracellular expression of biologically Streptokinase in E.coli. In order to express native-like, full S. equisimilis strain H46AStreptokinase intracellularly in E. coli the SK-encoding polynucleotideblock was transferred from the plasmid vector construct pJKD-55 bydigesting with Nco I and Sal I restriction enzymes (R.E.) whichliberated the SK open-reading-frame (ORF). Plasmid pJKD-55 contained thestreptokinase gene which was isolated from Streptococcus sp. (ATCC12449), also referred to conventionally in the scientific literature asS. equisimilis strain H46A, by known procedure earlier reported for themolecular cloning of SK gene and its expression in heterologous hostssuch as E. coli (Malke, H. and Ferretti, J. J., 1984; Proc. Nat'l Acad.Sci. 81: 3557; Pratap, I., Kaur, J., Rajamohan, G., Singh, D., andDikshit, K. L., 1996; Biochem. Biophys. Res. Commun. 227: 303). Thelatter publication describes the procedures by which the SK gene wascloned in E. coli plasmids, such as pJKD-8 and pJKD-55 used herein (seebelow).

The DNA segment liberated from pJKD-55 by R. E. digestion was thencloned into plasmid pET-23(d) (see FIG. 8 for map of this plasmid) whichhad also been treated with the same enzymes (Nco I and Sal I) to obtaincohesive ends compatible with those of the SK gene (see FIG. 9 for thescheme used for this purpose). This vector contained an initiation codonin-frame with the Nco I site of pET-23(d). Upon ligation, the SKopen-reading-frame could be recreated, but one modified at theN-terminal end, together with an additional ATG at the 5′ end emanatingfrom the re-formed Nco I site.

The construction of pET23(d)-SK was carried out as follows. Approx. 3 ugeach of pET23(d) and pJKD-55 plasmid DNAs were digested (separately)with 20 units each of Sal I (37° C. for 6 h), followed by 15 units eachof Nco I in 20 ul reactions at 37° C. for 10 h. After stopping thereactions by heat treatment (65° C., 10 min), followed byphenol-chloroform extraction and ethanol precipitation of the DNA, thedigests were run electrophoretically on a 1.2% agarose gel to isolatethe needed DNA fragments i.e. insert, carrying the SK gene from pJKD-55,and the linearized vector pET23(d) [see FIG. 9]. The respectivefragments were purified from the gels using the Prep-A-Gene DNApurification kit of BioRad Inc., CA, USA. The insert anddouble-digested, linearized vector DNAs were then ligated at an approx.3:1 molar ratio (.about.350 ng of vector and 400 ng of the insertliberated from pJKD-55) in a 20-uL reaction using standard ligationconditions at 16° C. for 12 h. After this duration, the ligase was heatinactivated (60° C., 15 min) and one-fifth of the ligation reaction wasdirectly used to transform E. coli XL-Blue electrocompetent cells usingthe following electroporation conditions with 2 mm internal diameterelectroporation cuvettes (obtained from BioRad Inc., Richmond, Calif.,USA): voltage, 2.5 KV; resistance, 200 Ohms, and capacitance 25 uF. Sixtransformants were picked up from Amp-LB plates on which the transformedcells were plated at various dilutions. Individual colonies wereinoculated into 10 ml LB-Amp media to prepare plasmid DNA by standardmethods. The isolated plasmids were then screened electrophoretically onthe basis of molecular size to identify the positive clone/s. All sixclones were positive by this criterion. In order to express full-lengthSK containing all of the amino acid residues of mature S. equismilis SK(FIG. 3), the native N-terminal was repaired using a synthetic“cassette” approach (refer to FIG. 10 for the scheme followed for therepair of the SK gene). The portion of the repaired SK gene at the 5′end in pE13(d)SK was obtained through PCR using the primers RG-6 andRG-7 with the following sequence and target specificity.

RG-7 (forward primer) 5′-ATT GCT GGA CCT GAG TGG CT-3′ (SEQ ID NO:25)(specific for the first seven codons of the SK gene; SK FIG. 11) RG-6(reverse primer) 5′-TGG TTT TGA TTT TGG ACT-3′ (SEQ ID NO:26) (specificfor codons 57-62 of SK gene)

The PCR was carried out using as the template, plasmid pSKMG-400, whichcontained the DNA sequences coding for full-length native SK ofStreptococcus sp. (ATCC 12449), also referred to as S. equisimilis H46Aas described earlier. This plasmid was constructed by cloning the NcoI-Sal I fragment obtained from pJKD-55 followed by T4 DNApolymerase-catalyzed fill-in of the two ends (to obtain blunt ends) andcloning at the Eco RV site of plasmid BlueScript KS⁻. (Stratagene Inc.,WI, USA). The two PCR primers were designed to amplify the N-terminalportion of the native SK gene upto a unique restriction site in the genewhich could be utilized for recloning the amplified PCR product backinto pET23(d)-SK for expression of protein. Moreover, the 5′ end of theRG-7 primer started with ATT, coding for Ile, the first residue of themature SK-encoding DNA (or gene), so that the PCR-amplified SK-encodingpolynucleotide DNA segment could dock in-frame with the nucleotides ATG,formed at the NcoI-cut and refilled end of the expression vector, thusjuxtaposing the initiation codon in-frame for the repaired SK ORF. Thefollowing PCR conditions were used for the amplification reaction (100ul total): approx. 10 ng pSKMG-400 as template, 20 pmol each of the RG-6and RG-7 primers, 1 ul (2.5 units) of pfu DNA polymerase (StratageneInc.), 200 uM of each dNTP's, 10 uL of the standard buffet (10× conc.provided by the Stratagene Inc.). The following cycling parameter wereused: ‘hot start’ for 5 minutes at 92° C., denaturation at 92° C. for 1min, annealing at 50° C. for 1 min and extension at 72° C. for 1 min. Atotal number of 30 cycles, and a final extension of 10 min at 72° C. forallowing the completion of any of the incomplete amplified products,were provided. The PCR showed a single band of 160 bp as evidenced byelectrophoresis on a 1.2% agarose gel. For cloning the PCR product intopET23(d)-SK vector, approx. 10 ug pET23(d)-SK vector was digested with25 units of Ncol restriction enzyme in a 100 ul reaction using thebuffer (NEB-4) supplied by New England Biolabs, Inc., and by incubatingat 37° C. for 6 h. The completion of Ncol digestion was checked byloading 5 ul of the reaction mixture on a 0.7% agarose gel. Afterconfirming the digestion, the NcoI site was filled-in (i.e. made bluntended) using T4 DNA polymerase in the presence of all four dNTPs in a85-ul reaction as follows. Seventy five ul of above-mentioned Nco Idigestion mixture was supplemented with 4 ul DTT (100 mM stock). 4 uldNTP's from a dNTP stock (2 mM), and 2000 Weiss units of T4 DNApolymerase. The reaction was incubated at 37° C. for 1 h after which itwas stopped by adding EDTA (10 mM final conc.) and heating at 75° C. for10 min. The DNA was then ethanol-precipitated. The precipitated DNA wasdissolved in 40 ul TE and was digested with Afl II restriction enzyme ina 60-ul reaction at appropriate reaction conditions as recommended bythe supplier. Separately, 40 ul of the PCR-amplified DNA reaction,prepared using pSKMG-400 vector as substrate to supplement the deletedportion of the SK gene, was also digested with Afl II restrictionenzyme, followed by running on low melting agarose gel (1%) to separatethe vector and inset DNA pieces [the insert contained a blunt end, andan Afl II-site compatible cohesive terminus at the other end, thusmaking it suitable for facile ligation with the vector, which had beensimilarly treated with Nco I, followed by a fill-in reaction with T4 DNApolymerase to obtain a blunt end, followed by a digestion with Afl II].The required pieces of DNA were isolated from the electrophoresis gelsas small agar blocks after visualization under trans-illuminated UVradiation, and were purified from the agarose by beta-agarase enzyme.One unit of beta-agarase per 100 ul of agarose gel approximately in the1× beta-agarose buffer (New England Biolabs Inc.) was employed to digestthe agarose and to purify the DNA according to the protocol recommendedby the supplier (New England Biolabs Inc., USA). The purified DNAs werequantitated and vector and insert were ligated in a 1:5 molar ratio in a20 ul reaction, carried out at 16° C. for approx. 18 h. For the ligationreaction, 2 ul of 10× ligase buffer, 1 ul of 10 mM rATP stock, and 2000Weiss units of T4 DNA ligase were used. The DNA from the ligationmixture was precipitated with n-butanol, and used directly to transformelectrocompetent E. coli XL-Blue cells. The transformation mixture wasplated on LB-Amp plates. The positive clones (repaired pET23(d)SK) werescreened from the wild-type background on the basis of Nco I digestion(the insertion of the PCR amplified segment in the vector would resultin the loss of the Nco I site). Two of the clones (pETSK-NTRN, for‘N-terminally repaired, native’) obtained after this screening werefurther confirmed using Sanger's method of nucleotide sequencing; whichshowed complete fidelity with the known full-length native sequence ofthe S. equisimils SK-encoding DNA (Cf. FIG. 11) except the presence ofan extra ATG codon at the 5′-end of the ORF, and no mutation/alterationat the upstream promoter regions or downstream sequences in the plasmidcould be observed. DNA from these confirmed clones were then transformedinto E. coli BL-21 strain, and expression of intracellular SK in liquidculture was examined after induction with IPTG according to the protocoldescribed earlier, essentially by analyses of cell-lysates on SDS-PAGE.However, no band corresponding to standard SK was visible on SDS-PAGE.The possibility of the presence of low levels of SK was then checked byWestern Blotting analysis of the lysates as it is a more sensitivemethod when compared to a direct examination of the SDS-PAGE gels byCoomassie staining. In this case, indeed a faint band corresponding tothe position of standard SK on the Western blots could be clearlydiscerned, which showed that the levels of expression of the native SKgone was poor.

The possibility that the sequences in the native SK-encoding DNApolynucleotide block corresponding to the N-terminal residues could beforming strong secondary structure/s in the encoded mRNA transcriptsthat might be hindering the expression was through computer-assistedanalysis using the program DNASIS (version 5.0). This unambiguouslydemonstrated that the potential for forming highly stable secondarystructure by the N-terminal end of the SK gene was appreciably strong(free energy approximately −10 Kcal/mol; see FIG. 12A). Translationallysilent mutagenesis of the genes at its 5′ end was then carried out todisrupt and/or reduce this secondary structure by replacement of GC richcodons (that are more likely to promote secondary structure-formation inmRNA transcript) with AT-rich codons, wherever possible. Through thisprocedure several sequence/s, altered specifically at the 5′-end andpossessing lowered stability (−6 to −5 Kcal/mol) as compared to that ofthe native sequence, were obtained. One of these sequences, thatresulted in maximal destabilisation of structure-forming potential, toapprox. 5 Kcal/mole (FIG. 12B), was chosen for the expression studies.

The preparation of an expression vector containing a full-length, nativeSK-encoding polynucleotide segment but one with a modified (i.e.non-native) DNA sequence at its 5′-end with less structure-formingpotential was carried out as shown schematically in FIG. 13 using acombination of synthetic DNA cassette incorporation and PCR-basedstrategy. The alternate sequence to be incorporated at 5′-end of the SKgene was provided through two homologous synthetic oligonucleotides(SC-I and SC-II, complementary to each other except for overhangs at theend), whose sequence is shown below. Also indicated in bold type are thealtered nucleotides which resulted in a lowering of structure formingpotential in the 5′-end of the SK gene.

SC-I 5′-C ATG ATA GCT GGT CCT GAA TGG CTA CTA GAT CGT CCT TCT GTA AATAAC AGC C-3′ (Partial NcoI site) SC-I 5′-AA TTG GCT GTT ATT TAC AGA AGGACG ATC TAG TAG CCA TTC AGG ACC AGC TAT-3′ (Partial Mfe I site)

These carried two new restriction sites (NcoI and Mfel), introduced bysilent mutagenesis using the computer program GMAP (Cf. Raghava andSahni, 1994:, BioTechniques. 16: 1116-1123) without altering the aminoacid sequence encoded by this segment of DNA so as to facilitate thecloning of the repaired SK-encoding DNA into the expression vector,pET23(d)-SK (see FIG. 13 for the overall cloning scheme forreconstruction of the N-terminal region of S. equisimilis SK-encodingDNA). The alterations were carried out in two stages, as depictedschematically in FIG. 14. In Stage I, a translationally silentrestriction site (Mfe I) was engineered close to the N-terminal end ofthe SK gene (overlapping condon numbers 17 and 18 in the native SKsequence; see FIG. 3) since no unique site close to the N-terminal endwas available for incorporating a synthetic DNA piece for purposes ofaltering this region in the plasmid. An upstream PCR primer (termed ‘MfeI primer’) incorporating this potential Mfe I site (underlined in thesequence of the primer) was synthesized with the following sequence.

Mfe I primer: 5′-C-AGC-CAA-TTG-GTT-GTT-AGC-GTT-GCT-3′

A synthetic oligonucleotide containing an Afl II site (termed RG-6,which has been described before) was used as the downstream primer.

These two primers were utilized for the amplification of the SKsequences encoding the N-terminal region using pfu DNA polymerase. Thefollowing reaction conditions and cycling parameters were used. Pfupolymerase buffer (Stratgene Inc.), 200 uM each of the dNTP's, MfeI andRG-6 primers: 20 pmol each. pET23(d)-SK vector as template (2 ng), Pfupolymerase 1 ul (2.5 units), total reaction volume 100 ul. A ‘hot start’was given for 5 min at 95° C., followed by denaturation for 1 min at 95°C., annealing for 1 at 45° C., and extension for 1 min at 72° C. A finalextension at 72° C. for 10 min was also incorporated in the program. Asexpected from theoretical considerations, a 14]-bp long SK region wasamplified. The PCR product was phenol-chloroform purified andprecipitated using isopropanol after adjusting the salt concentration to0.3 M with 3 M NaOAC. The precipitated product was dissolved in 25 ulsterile dist. water and kinase in a 30-ul reaction, after adding 3 ulMulticore buffer (Promega Inc., WI. USA), 1 ul (10 units) of T4 PNK(Promega) and 1 ul rATP (10 mM stock). The reaction mixture wasincubated at 37° C. for 2 h and then stopped by heat-inactivating at 65°C. for 20 min, and the DNA purified using phenol-chloroform andprecipitated with 2 volumes of isopropanol. The pBluescript 1 KS(−)vector was digested with EcoRV restriction enzyme and thendephosphorylated using CIAP using a standard protocol. Both the kinasePCR product and dephosphorylated pBluescript II vector were quantitatedby A₂₆₀ measurements in a 100-uL cuvette, and the vector and insert DNAswere ligated in 1:10 molar ratio of vector: insert by taking 590 ngvector and 280 ng insert in a 20 ul reaction after adding appropriateamount of ligase enzyme (approx. 500 Weiss units) and ligase buffercontaining rATP. The ligation reaction was incubated at 16° C.overnight. The ligation mixture was heat inactivated (65° C., 30 min),the DNA was butanol-precipitated, and approx. one-fifth electroporatedinto E. coli XL-Blue electrocompetent cells. The transformants werescreened by plating them on LB-Amp plates. Ten transformants were pickedup and inoculated for minipreparation of plasmid DNA. The minipreps werethen digested with MfeI and AflII enzymes sequentially to identify thepositive clones containing the 141 bp insert. Unmodified pBluescript waskept as control. All the transformants were found to be positive by thiscriterion. This construct was labelled as p(MfeI-AflII)-SK.

Stage-II: The oligos SC-I and SC-II in equimolar amounts (approx. 270 ngeach) in 25 uL were annealed by cooling their mixture from 80° C. toroom temperature slowly. Approximately 5 ug of pET 23(d)SK and 10 ugp(MfeI-AflII)-SK vectors were digested with Afl II/Nco I and Mfel/AflII,respectively, for vector and insert preparations. Twenty five units eachof MfeI and AflII were used for vector preparation and 50 units wereused for insert preparation. The enzymes were added in two shots of 12.5units and 25 units in each of the reactions. The pET23(d)-SK vector wasdigested in a 60 ul reaction, and p(MfeI-AflII)SK was digested in a100-ul reaction. The pET23(d)-SK digestion mixture was run on a 1% lowmelting agarose gel for vector preparation and the p(MfeI-AflII) SKdigestion mixture was run on a 2% agarose gel for isolating the 115 bpinsert. Both the vectors and insert bands were cut out from the agarosegel and were purified using beta-agarase and quantitated. Then, amixture of Nco I and Afl II digested pET23(d)-SK vector, annealed SC-Iand SC-II oligos, and Mfe I-Afl II insert of p(Mfe I-Afl II)-SK vectorwere ligated in a 3-piece ligation reaction in a 1:7:5 molar ratio in a20 ul reaction (see FIG. 13). In the actual reaction, approx. 660 ng ofthe vector, 92 ng of the insert and 60 ng of the annealed oligos wetaken. The mixture was ligated by adding 2000 Weiss units of ligase intothe reaction. The reaction was incubated at 16° C. overnight. Theligation mixture was n-butanol-precipitated, dried, redissolved in 10 uLTE and approx. one-third used to transform E. coli XL-1 Blueelectrocompetent cells. The transformants were screened on LB-Ampplates, Ten transformants were picked and inoculated for preparation ofminipreps. All the minipreps along with pET23(d)-SK as control weredigested with Nco I enzyme to search for the positive clones. Only oneclone, as well as pET23(d)-SK, gave digestion with Nco I which indicatedthat the remaining 9 clones were positive for the desired construct. Oneof the clones was then completely sequenced by automated DNA sequencingusing Sanger's dideoxy method, which showed that the N-terminal regionwas now full-length i.e. encoded the native SK sequence plus aN-terminal methionine, containing exactly the sequence expected on thebasis of the designed primers, SC-I and SC-II with the altered codons atthe 5′ end compatible with potential for secondary structure reductionin the mRNA transcripts (see FIG. 14). In addition, the DNA sequencingestablished that no other mutation was inadvertently introduced in theSK ORF in during the reconstruction protocol. This plasmidvector-construct, termed as pET23(d) SK-NTR (N-terminally reconstructed)has been deposited in the Microbial Type Culture Collection, Chandigarh,India (MTCC), with the accession No. BPL 0017. The plasmid DNA from thisclone was then transformed into E. coli BL-21 DE3 strain for expressionstudies. The E. coli BL-21 cells were grown in liquid culture andinduced with 2 mM IPTG at an OD₆₀₀ of ˜0.6 for the induction of SK, asdetailed earlier. The cells were then pelleted by centrifugation andlysed in SDS-PAGE sample buffer and analysed electrophoretically bySDS-PAGE on 10% acrylamide gel. It was observed at the level of SK (47kD band) was approx. 25-30 percent of the total soluble proteins, asubstantial increase compared to the very low expression observed in thecase of the construct with the native N-terminus (pET 23(d)-SK-NTRN).

Example 2

Harvesting of intracellularly expressed SK from E. coli, purification ofSK protein, and characterisation of highly pure and biologically activeSK.

Glycerol stocks of E. coli BL-21 strain harbouring plasmidpET23(d)SK-NTR, maintained at −70 C, were used to prepare a seed cultureby inoculating freshly thawed glycerol stock (approx. 100 uL) into 100ml of LB medium (in a 500 ml conical flask) containing 50 ug/ml ofampicillin. The flask was incubated at 37° C. with shaking on a rotaryshaker at 200 r.p.m. for 16 h. This culture was used to seed four 2 LErl nmeyer flasks each containing 500 ml of the same medium (LB-Amp)using 5% (v/v) of inoculum. The flasks were incubated at 37° C. withshaking (200 r.p.m.) for a duration till the absorbance at 600 nm hadreached 0.5-0.6 (.about.2 h after inoculation). At this time, IPTG wasadded to the cultures to a final conc. of 2 mM and incubation, asbefore, continued for a further 3 h. The cultures were then chilled onice and processed for the next step immediately. The cells from theculture media were harvested by spinning them down by centrifugation at6000×g in a GS-3 rotor (Servall) for 30 min at 4° C. The supernatantswere discarded and the combined cell-pellets carefully resuspended byvortexing in 65 ml of lysis buffer containing a chaotropic agent foreffecting release of the cellular contents. The composition of the celllysis buffer was as follows (final concentrations are given): 6 Mguanidine hydrochloride and 20 mM sodium phosphate buffer, pH 7.2.

The E. coli cell suspension was shaken gently on a rotary shaker at 4°C. for 1 h to effect complete cell lysis. The lysate was then subjectedto centrifugation at 4° C. for 15 min at 9000 r.p.m. The clearsupernatant (containing approx 300 mg total proteins as determined bythe Bradford method) was then processed further, as follows (allsubsequent steps were conducted at 4° C., and all buffers and othersolutions used were also maintained at 4° C.). The supernatant wasdiluted 6-fold in which the conc. of Gdn.HCl was 1 M; simultaneously,aliquots of a stock solution (0.5 M) of sodium phosphate buffer, pH 7.2,n NaCl (stock conc. 5 M) were added to obtain 20 mM and 0.5 M withrespect to sodium phosphate and NaCl, respectively, in the diluted celllysate supernatant (final volume 200 ml). The mixture was gently swirledfor a few minutes, and then loaded onto a 100 ml bed volume (4 cminternal diameter) axial glass column for hydrophobic interactionchromatography (HIC) on phenyl-agarose-6 XL (Affinity ChromatographyLtd., Isle of Main, U.K.) coupled with an automated liquidchromatography work-station (model Biocad Sprint, Perseptive Biosystems,Mass., USA) capable of continuous monitoring of effluents at twowavelengths simultaneously, and formation of predefined gradients forelution. The column was pre-equilibrated width 0.5 M NaCl in 20 mMsodium phosphate buffer, pH 7.2 (running buffer) onto which thebacterial cell lysate was loaded at a flow rate of 85 ml/h. Theflow-through was collected, and the column washed with running buffer(400 ml total) at the same flow rate, followed by the same volume ofrunning buffer devoid of NaCl (washing steps). The SK was then elutedwith dist. water (pH 7.0) at a slower flow rate (35 ml/h). All theeffluents were collected in fractions (25 ml each) and the SK activityas well as protein content in each fraction was determined. Virtuallyall of the loaded SK activity was found to bind to the column, less than5% of the total activity being found in the flow-through and washings.Approximately 85-90% of the loaded SK activity was recovered at thedist. water elution step. SDS-PAGE analysis showed the presence of apredominant band of 47 kD migrating alongwith native SK (purified fromS. equisimilis H46A) run as standard. The SDS-PAGE as well as theactivity analysis showed the SK to be 85-90% pure at this stage whencompared to the unpurified cell lysate. The SK in the dist. water elutewas then made 26 mM in sodium phosphate, pH 7.2 (running buffer) andloaded at a flow rate of approximately 300 ml/h onto DEAE-Sepharase FastFlow (Pharmacia, Uppsala, Sweden) packed in a 1.6×20 cm axial glasscolumn pre-equilibrated with the running buffer. The column was thenwashed with 200 ml of the same buffer, following which it was developedwith a NaCl gradient (0-0.6 M) in running buffer (pH 7.2). All eluatesfrom the column were saved with an automated fraction collector. Ten-mlfractions were collected, and SK activity as well as protein wasestimated in each. Aliquots from each fraction were also analysed bySDS-PAGE to examine the relative purity of the eluted protein. Theflow-through and washings were essentially devoid of SK activity, butapproximately 80-85% of the loaded SK activity eluded at around 0.35 MNaCl in the gradient as a single symmetrical peak (containing a total of42 mg protein). The specific activity of this protein was 1.1×10⁵I.U./mg. On SDS-PAGE, it showed a single band co-migrating with standardnatural S. equisimilis SK. A densitometric analysis of the SDS-PAGE gelsrevealed that the background protein/s in the final purified SKrepresented less than 2% of the total Coomassie stainable content. Theoverall SK recovery with the purification process was found to beapprox. 65 percent.

The purified recombinant SK expressed in E. coli was characterizedphysico-chemically by several other criteria in order to compare it withnatural SK. By the clot lysis procedure, it showed a specific activityof 105,000 IU/mg, under conditions where natural SK from S. equisimilisstrain H46A was found to have a specific activity of 110,000 IU/mgprotein. Upon reverse phase high performance liquid chromatography(RP-PLC) on C-18 columns, both SK types were indistinguishable, showingthe presence of a single symmetrical peak at the same position wheneluted with a gradient of gradually increasing ACN concentration. By UVspectroscopy, the recombinant SK was found to be identical to thenatural SK. The N-terminal amino acid sequence of rSK was found to beidentical with that of natural SK, except for the presence of an extramethionine residue at the N-terminus (the sequencing was carried out for25 cycles).

Example 3

Construction of a hybrid DNA polynucleotide between SK-encoding DNA andfibrin binding domains 4 and 5 encoding DNA of human fibronectin, itsexpression in E. coli, oxidative refolding, and purification ofbiologically active chimeric protein.

The scheme followed for the construction and expression of a chimeric(hybrid) polynucleotide DNA block formed between the DNA encoding forresidues 1 to 383 of SK followed by in-frame joining to the DNA codingfor the FBD 4 and 5 of human fibronectin is shown in FIG. 16. A shortlinker DNA segment, coding for 3 glycine residues, in tandem, betweenthe two polynucleotide-segments was incorporated into the design (termed‘intergenic sequence’) (see FIG. 15) so as to provide flexibility to theexpressed chimeric polypeptide product. In addition, a new terminatorcodon was introduced at the end of the FBD(4,5) DNA so that the hybridORF encoded for a polypeptide ending after the two FBDs. Thus, thedesign essentially had the following configuration: SK[residues1-383]-(gly-gly-gly)-FBD(4,5)]. In addition, a transglutaminaserecognition site was also engineered in the gene-design directly afterthe intergenic sequence so that the expressed, hybrid protein couldbecome covalently cross-linked to the fibrin strands of the clot (FIG.15). A two-stage PCR-based experimental strategy (FIG. 16) was employedto construct the hybrid polynucleotide. A polynucleotide-blockcontaining the sequence coding for domains 4 and 5 was first selectivelyamplified using the plasmid pFHMG-60 as template. The latter containedthe DNA encoding for all five human FBDs (FIG. 16). This amplificationreaction (PCR-I) was carried out with specifically designed forward andreverse primers with the following sequences.

Forward primer (MY 13): 5-CCG GAA TTC GCG CAA CAG ATT GTA CCC ATA GCTGAG AAG TGT TTT GA-3′ Eco RI Transglutaminase- hybridizes to upstreamrecognition sequence FBD (4, 5) sequences Reverse primer (MY 14): (SEQID NO:17) 5′-GGC CTT AAG AGC GCT CTA ACG AAC ATC GGT GAA GGG GCG TCTA-3′ ‘clamp’ Afl II Eco 47 III hybridizes to downstream stop codon codonFBD(4, 5) sequencesNote:— In the above primer sequences, the 5′-non-hybridising sequences(bold) as well as the hybridizing ones, towards the 3′-ends of theprimers that are complementary to selected segments of fibronectinFBD(4,5)-encoding DNA sequences are shown. In the 5′-non-hybridizingends were also incorporated new R.E. sites by ‘silent’ mutagenesis, atransglutaminase(TG)-encoding site and/or stop codon sequences, asindicated above (underlined). The start of the hybridizing sequences #in primer MY-13 correspond to the beginning of the sense strand sequenceof FBD(4,5), namely residue 150 onwards (refer to FIG. 6 for the aminoacid and DNA sequences of the fibrin binding domains of humanfibronectin). In case of primer MY-14, the beginning of the hybridizingsequence (antisense) correspond exactly to the codon for residue 259 ofhuman fibronectin (Cf. FIG. 6). The ‘clamp’ mentioned in the figurerefers to the extra nucleotides added at the 5′ end of a # primer tofacilitate the digestion at the nearby R.E. site which, otherwise, ispoorly digested when present at or near the end of a DNA fragmentgenerated by PCR.

As described above, the forward primer contained a segment at its 3′ endthat was homologous with the 5′ end of the DNA encoding for the FBD(453)sequences, and also contained a 5′ (nonhybridizing) segment that encodedfor a TG-recognition site is well as RE sites to facilitate the cloningof the PCR product in a plasmid vector. This plasmid, containing theFBD(4,5) gene-block and additional 5′ sequences was then employed astemplate for a second PCR (PCR-II) using a set of primers (RG-3 andMY-14). Primer RG-3 was designed so as to contain the other desiredelements of the intergenic segment viz, the codons for the gly-gly-glyresidues, as well as those encoding for a small segment of SK (see FIGS.15 and 16) directly after a unique R. E. site (Bsm I) present naturallyin the C-terminal region of the native SK ORF (approximately overlappingthe codon 377 and 378 of the S. equismilis SK open-reading-frame; Cf.FIGS. 3 and 4). This site was chosen as the common, junction-pointbetween the two polynucleotide blocks (i.e. SK and FBD) to beintegrated. Additionally, unique restriction sites flanking theintergenic (i.e. in and around the -gly-gly-gly-) sequences were alsodesigned into the upstream primer through translationally silentmutagenesis that could be exploited to substitute alternateoligonucleotide cassettes at the intergenic region of the hybridpolynucleotide that would provide altered flexibility and/or rigiditycharacteristics in the expressed polypeptide different from thatprovided by the (gly)₃ linker (Cf. FIG. 15).

The sequence of primer RG-3 is given below highlighting featuresincorporated in its design (bold letters denote non-hybridizing segmentstowards the 5′-end of the primer to distinguish these from the sequencecomplementary with respect to template DNA).

(SEQ ID NO:18) 5 5′-G AAT GCT AGC TAC CAT TTA GCT GGT GGT GGC CAG GCGCAA CAG ATT GTA CCC-3′ Bsm I Bst X Xcm I Bal I segment hybridizing with(hybridizes to SK the 5′-end of DNA block gene at codons (-gly-gly-gly-)from PCR-1 at the TG 376-383) recognition site

The amplified DNA obtained from PCR-II using primers RG-3 and MY-14 wastreated with Bsm I to “dock” it at the Bsm I site in the SK ORF invector pSKMG400 at one end and with Eco 47 III (which producesblunt-ends) to facilitate blunt-end cloning at the filled-in Bam HI sitepresent after the SK open-reading-frame (ORF) in the plasmid vector(FIG. 16).

The following reaction conditions and PCR; parameters were used. PCR-I(final reaction conditions in a 100-uL reaction). 20 pmol of each of theMY-13 and MY-14 primers, pFHMG-60 vector as template (1 ng), 200 uM eachof the dNTP's, Pfu polymerase 1 ul (2.5 units). Pfu polymerase was addedat 94° C. i.e. a hot start for 5 min was given to avoid non-specificamplification. The following cycling conditions were employed:denaturation at 92° C. for 45 seconds, followed by annealing at 60° C.for 1 min, and extension at 72° C. for 1 min. A total number of 30cycles were given, followed by a final extension for 10 min at 72° C.The reaction yielded a single 360 bp PCR product as seen on a 2% agarosegel alongwith standard PCR markers. The amplified product was thencloned into pBluescript II KS⁻ at the EcoRI site (refer to FIG. 16)which had been introduced in the PCR product as a 5′-overhang. The PCRreaction mixture was purified using standard methods, and then kinasewith T4 PNK enzyme. The kinased PCR product was ligated to concatamarizethe PCR product in order to internalize the EcoRI site to facilitatecloning of the DNA using the procedure of Jung et al. (Jung, V., PestkaS. B., and Pestka, S. 1990, Nucl. Acids Res. 18:6156). The ligated DNAmixture was then digested with EcoRI followed by electrophoresis on a 2%agarose gel to check the efficiency of the ligation and digestion steps.The band representing EcoRI-digested PCR product was cut out form thegel and purified. Approximately 400 ng of the PCR product was ligatedwith lug of pBluescriptII (KS⁻) predigested with EcoRI and thendephosphorylated. Approximately 2 ul of the ligation mixture wasdirectly used to transform E. coli DH 5-alpha electrocompetent cells.The transformants were selected on LB-Amp plates containing IPTG andX-gal using blue and white colony selection. Ten white transformantswere picked and taken up for minipreparation of plasmid. The plasmidDNAs were digested with EcoRI enzyme, and the digests analysed on 1.5%agarose gels. The transformants releasing 360-hp insert (the size of thePCR product) were taken as positive clones. Nine of the ten clones werefound to be positive by this criterion. One of the above clones was thenconfirmed for the absence of any unexpected mutations by automated DNAsequencing by Sanger's procedure, and used as template for PCR-IIemploying RG-3 and MY-14 as primers so as to add additional sequences atthe 5′ end of amplified FBD sequences in the cloned PCR-I product. ThePCR-I had resulted in the addition of a TG recognition site and a stopcodon onto the original FBD(4,5) gene-block, whereas the stage-II PCRwas carried out to effect the addition of the poly-glycine linker andoverlapping SK sequences onto the FBD(4,5) gene-block. The followingreaction conditions and cycling parameters were used for carrying outPCR-II. Each of the dNTP's: 200 uM, PCR-I product cloned in pBluescriptII (KS⁻) as template (linearized) 260 ng, RG-3 and MY-14 as forward andreverse primers (100 pmol each), 5% [0140] DMSO (v/v), 1 ul Taq DNApolymerase (2.5 units) in a total reaction-volume of 100 ul. Cyclingparameters were similar to that of PCR-I except that the annealingtemperature was lowered to 58° C. An aliquot (approx. one-tenth) of thePCR-II was run on a 1.5% agarose gel to check for amplification. As theTaq polymerase does not produce blunt end PCR products (unlike pfupolymerase) but ones with a single-base overhangs (reference), the PCRproduct was first filled-in, and then kinased. These two modificationscarried out in a single reaction at 22° C. for 30 min using 10 unitseach of the T4 DNA polymerase and T4 PNK (total volume 85 ul). Inaddition, 8 mmol dNTP's as well as 1 mmol rATP were added to thereaction (all indicated concentrations are final). The reaction wasstopped by adding EDTA to 10 mM followed by heating the tube at 70° C.for 10 min. The filled and kinased PCR product was subjected to aphenol-chloroform treatment and precipitated with two volumes of ethanolin the presence of 0.3 M sodium acetate. The pellet was redissolved in20 ul of dist. water. Approx. 15 ul of this DNA was ligated in order toconcatemarize the PCR product. For doing the ligation, 1× UniversalBuffer (supplied by Stratgene Inc.), 1 ul (of a 10 mM rATP stock) and400 weiss units of T4 DNA ligase were added to a 25 ul reaction. Thereaction was incubated at 16° C. overnight. The ligase washeat-inactivated at 65° C. for 10 min. The concatamerized PCR productwas then first digested with Eco47 III (approx. 20 units) in a 25 ulreaction at 37 C for 6 h and then the DNA was digested with BsmI enzymeat 65° C. for 6 h after adding .about.20 units of BsmI enzyme in thesame reaction. In parallel, the vector pSKMG-400 (approx. 4 ug),containing the SK gene, was digested width BamHI enzyme according tostandard protocol and the digested DNA was filed-in using T4 DNApolymerase in the presence of 100 uM dNTP's and 0.5 mM DTT. The reactionwas incubated at 37° C. for 1 h. The reaction was stopped by heating thetube at 75° C. for 10 min. Then the BamHI filled vector was digestedwith BsmI enzyme by incubating at 65° C. for 6 h. The vector waspurified by a phenol-chloroform treatment followed by achloroform-isoamyl extraction, followed by ethanol precipitation of theDNA. Then the Eco47 II and BsmI double-digested PCR product andBamHI-digested and filed-in plus BsmI-digested pSKMG-400 vector wereligated (refer to FIG. 16) in 20-ul reaction by incubating at 16° C. for14 h, after which the ligase was inactivated by heating at 70° C. for 10min and then the DNA was precipitated with n-butanol. It was then usedto transform E. coli XL-Blue electrocompetent cells. The transformedcolonies were then selected on LB-Amp plates. Ten transformants werepicked and screened for the presence of the diagnostic test, namely therelease of a 372-bp fragment after digestion with NotI and BamI enzymes,in contrast to a 295-bp fragment from the control plasmid, pSKMG400since the positive clones contained the additional FBD(4,5) segmentEight clones from ten selected turned out to be positive by thiscriterion. The positive clones were designated as pSKMG400-FBD(4,5). Oneof these was subjected to DNA sequencing which confirmed the presence ofthe expected sequence at the 5′-end, and a complete absence of any othermutation in the rest of the gene-block.

For the expression of the SK-FBD(4,5) hybrid polynucleotide DNAintracellularly in E. coli, the scheme shown in FIG. 17 a was followed.The BsmI-NotI DNA piece from pSKMG400-FBD4-5 [beginning from thatencoding for the C-terminal portion of the SK gene and carrying theintergenic linker region, the FBD(4,5) segments, and ending after thestop codon for this ORF at the Not I site originating from the multiplecloning site (MCS) of the parent vector] was transferred into plasmidpET3(d)SK-NTR digested with the same restriction enzymes. The digestedvector and the DNA segment from pSKMG400-FBD(4,5) (insert) were isolatedfrom 1% agarose gels after beta-agarase digestion, as described earlier,The vector and insert DNA were then ligated by standard procedures usinga vector insert molar ratio of 1:5 (approximately 590 ng of theBsmI/NotI double-digested vector and 250 ng of the BsmI/NotIdouble-digested insert in a 20-ul reaction). DNA from the ligationreaction was butanol precipitated and directly used to transform E. coliXL-1 Blue electro-competent cells. Transformants were selected on LB-Ampplates. Ten transformants were picked up and taken up for plasmidminipreparation. The miniprep DNAs were digested with BsmI and NotIenzymes, alongwith pSKMG400-FBD(4,5) as control. Three clones gaveBsmI/NotI insert equal in size to that of the insert liberated frompSKMG400-FBD(4,5). One of the clone, designated pET23(d)SK-NTR-FBD(4,5)and deposited with MTCC with accession No. BPL 0014, was then fullysequenced by automated DNA sequencing in and around the SK insert (seeFIG. 17 b for the gene sequence). All the 3 positive clones weretransformed into E. coli BL-21 strain and were induced for theexpression of SK-FBD(4,5) hybrid protein using the standard protocoldescribed before. The E. coli BL-21 cells harboring the plasmidpET-SK-FBD(4,5) were induced with 2 mM IPTG at .about.0.60D₆₀₀ and werefurther incubated for 3 h at 37° C. In parallel, cultures were alsogrown where IPTG addition was omitted (uninduced controls). Cells from1.5 ml of the cultures were pelleted down by centrifugation and weredirectly lysed in 100 ul SDS-PAGE sample buffer. After high-speedcentrifugation (8000 g×20 min) to pellet undissolved components, approx.25 ul of the supernatant of each of the samples (alongwith lysate frompET-23(d) SK-NTR, as positive control) was loaded onto 10% SDS-PAGE geland subjected to electrophoretic analysis. The gels showed distinctbands of 57 kD in the IPTG-induced cultures (roughly representing 20-25%of the total Coomassie-stained protein bands in the gel), indicatingthat the hybrid SK-FBD(4,5) fusion protein had been expressed at highlevels. In the case of pET23(d)SK-NTR harboring cultures, a bandcorresponding to 47 kD, the position of native SK, was observed. Inparallel, SDS-PAGE gels were subjected to the plasminogen-overlayprocedure, which showed distinct zones of clearance by the 57 kD hybridprotein, however, these zones were produced with a distinctly slowerrate in comparison to those produced by native SK or the rSK expressedin intracellularly from pET23(d)SK-NTR.

Ten ml of LB-Amp media were inoculated with E. coli BL21 cells harboringpET-SK-FN (4,5) and incubated at 37° C. for 12 h with shaking (200r.p.m.). This inoculum was used to seed 200 ml of LB-Amp medium, andincubated for 2 h at 37° C. with shaking. At this time, the OD₆₀₀ of theculture was approx. 0.600. The production of SK-FBD(4,5) protein in thisculture was then induced by the addition IPTG to 2 mM, and incubationcontinued for another 3 h at 37° C. with shaking. The OD₆₀₀ had reached1.2 by this time. The cells were harvested by centrifugation (8000 g×20min) at 4° C., washed once with ST buffer, and resuspended in approx 14ml of the same buffer over ice. This cell suspension was then subjectedto sonication in the cold (20 sec pulses with 20 sec. gaps; total time15 min). The lysate so obtained was then centrifuged (10000 g×30 min) at4° C. The supernatant was carefully decanted into a separate flask. Thissolution contained approx. 6 mg/ml protein as estimated by Bradford'smethod using BSA as standard, and had a total of 1.5×10⁴ I.U. of SKactivity as measure by the chromogenic peptide procedure (seedescription of methods, given above). The lysate was then split into twoportions of about 6 ml each to effect either air oxidation orglutathione-mediated oxidation of the SK-FBD(4,5) polypeptide. For theair oxidation, the 6 ml lysate was diluted to a total of 40 ml ofsolution (reaction mixture A) which contained (final concentrations):Tris-HCl (pH 7.5), 50 mM and NaCl, 150 mM. In reaction B (reoxidationusing the reduced-oxidized glutathione buffer), the final volume wasalso 40 ml, but it also contained [besides NaCl (150 mM) and Tris-Cl (50mM)], a mixture of GSSG and GSH (1:10 molar ratio, with GSH at 10 mM)and EDTA (1 mM). Both reactions were subjected to gentle mixing at roomtemperature (24 plus/minus 2° C.) for 10 min and then passed through twoseparate glass columns each containing 6 ml human fibrin-Sepharose at aflow rate of 20 ml/h in a recycled mode i.e. the effluent was passagedback into the column with the use of a peristaltic pump. After 18 h ofpassage through the respective columns, the flow of the reactionmixtures was terminated. Each column was then washed with 50 ml ofbinding buffer (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl) followed by 50ml each of 2 M urea (in binding buffer), followed by 6 M urea in thesame buffer (to effect tightly bound protein). All washings werecollected in 10 ml fractions with a fraction collector, and analyzed forprotein by Bradford's method, as well as SK activity using chromogenicsubstrate and human plasminogen as substrates was also determined foreach fraction. The total yield of protein in the 6 M urea washings was280 ug in the case of reaction A (air oxidation), and 380 ug in case ofReaction B (GSG-GSSH mediated refolding). These two pools represented,respectively, the fibrin-binding SK-FBD(4,5) protein obtained a airoxidation or glutathione-catalyzed refolding of the intracellularprotein expressed from the plasmidpET23(d)SK-FBD(4,5). The specificactivity of both the fibrin-Sepharose binding protein fractions fromReaction A and B were found to be almost identical (2.5×10⁴ IU/mg and2.1×10⁴ I.U./mg, respectively). The dilute protein factions wereconcentrated approx. 10-fold with centrifugation in Sartoriuscentristart filters. The concentrated fractions were then analyzed bySDS-PAGE, with and without reduction with beta-mercaptoethanol. Onreducing SDS-PAGE (i.e. with beta-mercaptoethanol treatment of thesamples), the reoxidized-refolded SK-(FN4,5), irrespective of the methodof reoxidation, showed a single predominant band, but one with higher MW(about.57 kD) as compared to the native SK standard (47 kD) as expectedon the basis of the chimeric design of the hybrid gene. Essentially thesame patterns were obtained when the SDS-PAGE was conducted withoutbeta-mercaptoethanol, a treatment wherein any of the cystine disulfide(S—S) bridges would not be reduced to cystine —SH groups. This indicatedthat the refolded molecules contained essentially monomericintra-molecular S—S bonds, and contained negligible quantities of highermolecular weight (i.e. polymeric) products formed due to inter-molecularS—S bridge formation between the SK (FN4,5) molecules as a result of thereoxidation step. When analyzed by the Ellman DTNB color reaction forthiol groups (Habeeb, A. F. S. A., 1972., Methods in Enzymol. 25:457.,Academic Press, New York) these fractions showed the complete absence ofany free —SH group, indicating that the oxidation of the original 8cysteines present in the reduced form of the chimeric polypeptides, toS—S bridges, was complete as a result of the reoxidation/refolding step.

Example 4

Construction of a hybrid DNA polynucleotide between DNA encoding for SKand FBD pair 1 and 2, and cloning and expression of the chimericpolypeptide in E. coli.

The construction of a hybrid between SK and FBD pair 1 and 2-encodingpolynucleotide DNA in the same translational frame involved a strategyclosely similar to that utilized for hybrid co construction between SK-and FBD pair 4 and 5-encoding polynucleotide DNAs (FIG. 18). Theessential units used in both the constructs were similar i.e., DNAencoding residues 1 to 383 of SK, a short polynucleotide sequenceencoding for polyglycine linker between the two DNA polynucleotideblocks, and a transglutaminase (TG) recognition site for cross-linking,removal of the stop codon of the SK gene and introduction of a new stopcodon at the end of the FBD segments [either FBD(4,5) or FBD(1,2) etc].This strategy also exploited the use of the Bsm I site of the SK gene asa common junction-point for the fusion between the SK and FBD(1,2)polynucleotide segments. However, the strategy differed from thatemployed for constructing SK-FBD(4,5) fusion in that the amplificationof the FBD (1,2) domains was carried out in one stage, unlike that ofSK-FBD(4,5) wherein two consecutive PCRs with differing 5′-primers wereutilized (Example 3). This was because in case of the SK-FBD(1,2)construct a very large primer was not required as a TG recognition siteis naturally present in the FN gene just at the beginning of the FBD-1domain (Cf. FIG. 6), thereby obviating the need to engineer a TG site inthe upstream primer.

The FBD pair (1,2) was amplified from the plasmid pFHMG-60 (containingall five of the FBD encoding sequences of human fibronectin) with thefollowing tow primers whose sequences are provided below. Also shown arethe 5′-ends containing non-hybridizing sequences (in bold letters) andthe incorporated RE sites therein to facilitate cloning and/or dockinginto the SK gene; the areas hybridizing with the target DNA sequences inthe templates are also underlined.

Upstream primer, MY-10 SK sequence (codons 377-3 83; Cf. FIG. 3) (SEQ IDNO:19) 5′-G-TAC-GGA-TCC G-AAT-GCT-AGC-TAT-CAT-TTA-GCG-GGT-GGT-GGT-CAG-GCG-CAG-CAA-ATG-GTT-3′ Bam HI Bsm I (-gly-gly-gly-) hybrdizes at thrTG-recogntn. site just before the FBD sequences Downstream primer, MY-6(SEQ ID NO:20) 5′-GGC-CTT-AAG-AGC-GCT-CTA-TTA-GAT-GGT-ACA-GCT-TAT-TCT-3′‘clamp’ Eco RI Eco 47 II Stop sequence hybridizing with FBD site        codons (1, 2) codons 99-104 (Cf FIG. 6)

The following conditions were used for the PCR: Each of the dNTP's: 200uM; pFHMG-60 vector as template: 1 ng; MY-10 and MY-6 primers: 20 pmoleach; pfu polymerase 2.5 units, 1× pfu polymerase buffer (StratageneInc.), and a total reaction volume of 100 ul. A ‘hot start’ was givenfor 5 min at 94° C. The following cycling parameters were useddenaturation at 92° C. for 45 sec, annealing at 50° C. for 60 sec, andextension at 72° C. for another 60 sec. A total of 30 cycles were given,after which a final extension was provided at 72° C. for 10 min. Theamplification of the SK-FBD (1,2) hybrid cassette was checked by loadingone-tenth of the reaction mixture on a 2% agarose gel. This demonstratedthat the expected DNA (369 bp) was satisfactorily amplified in theabsence of any background bands. The amplified PCR product was thencloned into pBluescriptII KS(−) after purification through QiagenPCR-purification column using the manufacturer's protocol. Approximately2 ug of pBluescriptII KS(−) vector was digested with 10 units of Sma Irestriction enzyme in 1×NEB-4 buffer in a 20-ul reaction by incubatingthe digestion mixture at 25° C. for 12 h. The enzyme was inactivated byheating at 55° C. for 5 min and the linearized plasmid DNA was purifiedafter electrophoresis on a 1% agarose gel. Then, 150 ng of theSmal-digtsted vector was ligated with approx. 120 ng of the purifiedinsert DNA (PCR product) in a 25-ul reaction after adding 2.5 ul of 10×(stock) ligase buffer (New England Biolabs Inc., MA, USA), 1 ul of 10 mMrATP stock and 400 weiss units of T4 DNA ligase. The ligation was doneby incubating the reaction tube at 16° C. for 12 h. After the ligation,the ligase was inactivated by heating at 70° C. for 10 min. The DNA inthe ligation mixture was precipitated with n-butanol and then dissolvedin 20 ul of dist. water and approx. one-third used to transform E. coliXL-Blue electrocompetent cells (Stratgene Inc., USA) by electroporation.Transformants were selected by plating on LB-Amp plates. Miniprepplasmid DNA was prepared from eight selected clones and analysed byagarose gel electrophoresis. The plasmid DNAs of the transformants wererun alongwith pure pBluescript II KS(−) to identify positive clones withlarger molecular weight (MW), signifying the presence of thePCR-generated inset DNA. Three transformants were found to be movingslower than the pBluescript DNA on 1.2% agarose eletsophoresis. Tofurther confirm that these contained the DNA insert, their plasmid DNAswere digested with EcoRI and BamHI enzymes since EcoRI and BamHI weretwo of the sites that were introduced in the PCR product duringamplification. This showed that a 370-bp fragment, corresponding to thesize of the PCR product was liberated, clearly establishing that theseclones contained the desired cassette. This was finally confirmed byautomated DNA sequencing by the Sanger dideoxy chain-termination methodwhich showed a complete correspondence with the sequence expected on thebasis of the primers and the target DNA viz, FBD(1,2) alongwith a shortstretch at its 5′-end carrying SK-specific and intergenic sequences. Thesequencing also established the absence of any other mutation in theamplified DNA. The cassette subcloned in pBluescriptII KS(−) was thentransferred into the SK-containing vector, pSKMG400, in order to fuse itin-frame with the SK ORF utilising the common Bsm I site. For cloningthe SK-FBD(1-2) hybrid cassette into pSKMG400 vector, both vector andinsert DNAs were first digested with BamI. Roughly 2 ug of thepBluescript-FBD(1,2) and 4 ug of the pSKMG400 were digested with 8 unitseach of the BamHI enzyme in a 30 ul reaction utilizing buffer D ofPromega. The tubes were incubated at 37° C. for 6 h. A small aliquot wasrun on a 0.7% agarose gel to check for the digestion. After confirmingcompletion of digestion the reaction was stopped by adding 0.1 volume of100 mM EDTA. The digested samples were loaded onto 0.8% agarose gel andthe desired fragments were cut out as agarose blocks. The DNA wasextracted by treatment with beta-agarase as detailed before, andquantitated. Ligation reaction was set up between double-digested vectorand the fragment containing the SK-FBD(1,2) cassette using 200 ng of thevector and 30 ng of the fragment, 4 ul of the ligase buffer, 4 ul of 10mM rATP, and .about.600 Weiss units of ligase in a total volume of 40 u.The ligation reaction was incubated at 16° C. for 12 h. The ligase wasinactivated by heating the tube at 70° C. for 10 min then the ligatedDNA was precipitated using n-butanol, air-dried and dissolved in a smallvolume of sterile distilled water. For the transformation step, approx.100 ng of the ligated DNA was used to transform E. coli XL-1 Blueelectrocompetent cells which were plated on LB-Amp plates. Five colonieswere picked up and used for plasmid minipreparations. The plasmid DNAswere digested with Afl II and Eco47 III restriction enzymes separately.pBluescript FBD(1,2) and pSKMG400 vectors digested with the same enzymeswere kept as controls. The digestion mixtures were run on a 0.7% agarosegel along with double-digested controls. Two clones show linearizationupon Eco47 III digestion. The pBluescript FBD(1,2) control also showedlinearization with Eco 47 III digestion, as expected. However, thepositive clones were of higher molecular size due to the presence of SK.The pSKMG400 did not show any digestion with Eco 47 III enzyme. Thepositive clones also gave out an insert upon AflII digested, asanticipated from the known presence of a single Afl II site SK andanother in the FBD(1,2) segment.

For the expression of the hybrid SK-FBD(1,2) polypeptide, the Bsm I-Not1 fragment from pSKMG400-FBD(1,2) was transferred into pET23(d)SK-NTR atthe same site (see FIG. 19 a). Approximately 10 ug of pSKMG400-FBD(1,2)plasmid DNA was digested with 15 U of Not I enzyme in NEB-3 buffersupplemented with 1×BSA by incubating at 37° C. for 6 h in a 60-ulreaction. A second addition of Not I enzyme was again made and thereaction mixture was further incubated for another 6 h. A small aliquotwas removed to check for the completion digestion by running an agarosegel. After the NotI digestion, the DNA was precipitated with ethanol andsod. acetate (0.3 M), redissolved in 20 ul of dist. water and digestedwith 20 U of BsmI enzyme in NEB-2 buffer in a 80-ul reaction at 65° C.for 14 h after overlaying the reaction mixture with 50 ul of mineral oilto avoid evaporation. Similarly, in parallel, approx. 5 ug of the vector(pET23(d)SK-NTR) was double-digested with 20 U of Not I and 15 U of BsmIenzymes, sequentially. The linearized vector, and insert were isolatedby running a 1% agarose gel and loading the above-mentioned digestionmixtures in well-separated wells. The vector and insert bands were cutout from the agarose gel using a clean scalpel and the respective DNAfragments were purified, quantitated spectrophotometrically and ligatedat a molar ratio of 1:5 of vector insert. Approximately 600 ng of theBsmI/NotI double-digested vector was ligated with around 250 ng of theBsmI/NotI double-digested insert in a 20-ul reaction by adding 600 Weissunits of ligase (NEB) and 2 ul of 10× ligase buffer (also of New Englandbiolabs, Inc.) and incubating for 14 h at 16° C. The ligation mix wasthen heat-inactivated at 70° C. and 15 min, and the DNA was n-butanolprecipitated, air-dried, dissolved in 20 uL of sterile water and approx.one-thirds directly used to electroporate E. coli XL-1 Blueelectrocompetent cells. The transformants were selected on LB-Ampplates. Ten transformants were picked up and inoculated into freshLB-Amp for plasmid minipreparation. The miniprep DNAs were digested withBsmI and NotI enzyme alongwith pSKMG400-FBD(1,2) as control. Threeclones were positive in terms of liberating an insert equal in size tothat of the insert liberated from pSKMG400-FBD(1,2). One of the cloneswas then sequenced to confirm the nucleotide sequence of the SK-FBD gene(see FIG. 19 b) and designated pET23(d)SK-NTR-FBD(1,2). This has beendeposited with MTCC under accession No. BPL 0014. The plasmid DNA forthis clone was transformed into E. coli BL-21 strain, grown in liquidculture and induced for the expression of SK-FBD(1,2) hybrid proteinwith IPTG, as described earlier. Cells from 1.5 ul of the inducedculture were pelleted down by centrifugation and were lysed in 100 ulmodified SDS-PAGE sample buffer; approx. 25 ul lysate, alongwith thatfrom cells harbouring pET23(d) SK-NTR as control was analysed on 10%SDS-PAGE gel. In parallel, cultures were also grown where IPTG additionwas omitted (uninduced controls) and similarly analysed alongwithinduced cultures by SDS-PAGE. The gels showed distinct bands of 57 kD inthe IPTG-induced cultures (roughly representing 20% of the solubleprotein fraction) indicating that the hybrid SK-FBD(1,2) fusion proteinhad been expressed at high levels intracellularly. In the case ofparallel pET3(d)SK-NTR harbor cultures, a major band corresponding to 47kD; the position of native SK was observed.

Example 5

In-frame fusion of DNA segments encoding FBD(4,5) at the N-terminal endof the open-reading-frame encoding for SK, and cloning and expression ofthe hybrid polynucleotide-construct FBD(4,5)-SK in E. coli.

The construction of the FBD(4,5)-SK hybrid polynucleotide DNA wasaccomplished by the splicing overlap extension (SOE) method, a procedurein which two (or more) DNA fragments are joined together employing PCRwithout using either DNA scission or ligation (in this context,reference may be made to the following publications. Horton, R. M.,Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R., 1989, Gene77:61). The two fragments to be joined by SOE need to have mutuallycomplementary sequences at their respective junctions where the joiningis to take place so as to form an ‘overlap’ (see FIG. 20). These regionsof complementarity can be engineered into the two DNA fragments, or‘blocks’, to be joined (in the present case, the SK and FBD sequences)through separate PCRs each employing primers specially designed for thispurpose. These two PCR-generated blocks are then used in the overlapextension reaction, in a third PCP, wherein the complementary strandshybridize partially at their 3′-ends through the regions of mutualcomplementarity after strand separation (denaturation) and reannealing.Thus, these two DNA strands act as megaprimers on each other, and in thepresence of thermostable DNA polymerase, the 3′-ends of thisintermediate are extended to form the full-length (i.e. fused) segment,which may then be further amplified using the flaking primers derivedfrom the first two PCRs used to generate the two DNA blocks. The FBD-SKpolynucleotide fusions were made using four synthetic oligonucleotidePCR primers viz., KRG-8, KRG-9, KRG-11 and KRG-12 whose sequences anddesign are described below (see also FIG. 19 for the overall schemefollowed for the construction of the chimeric gene-construct).

Upstream PCR-Iprimer KRG-8. Transglutamate recognition site                                  150     152     1545′-CC-ATG-GTG-CAA-GCA-CAA-CAG-ATT-GTA-CCC-ATA-GCT-GAG-AAG-TGT-3′ (SEQ IDNO:21) Partial Nco I hybridizes it begining of FBD(4) segment site(codon numbers of FBD are shown as per FIG. 6) Downstream PCR-I primerKRG-9. sequence complementary to codons 1-5 of SK (No.'s indicatedbelow) 5′-C TC-AGG-TCC-AGC-AAT- ACG-AAC-ATC-GGT-GAA-GGG-GCC-AGA-T-3′(SEQ ID NO:22)    5  4   3    2   1  259      257    255             253 sequencehybridizing with end of FBD(5) segment (No.'s indicated are codons, asper FIG. 6). Upstream PCR-H primer, KRG-11 FBD(5) sequence, as overhang;sequence hybridizing with SK gene; codon No.s Codon numbers (cf. FIG. 6)(Cf. FIG. 3) are indicated. are indicated. 5′-TTC-ACC-GAT-GTT-CGT-ATT-GCT-GGA-CCT-GAG-TGG-CTG-CTA-GAC-3′ (SEQ ID NO:23) 255        257259  1  3    5        7           9 Upstream PCR-II primer, KRG-125′-TGG-TTT-TGA-TTT-TGG-ACT-TAA-GCC-TTG-3′ (SEQ ID NO:24)   62       60  58       56     54 Note: sequence hybridizing with SKgene (codon No.'s are indicated; see FIG. 3)

As can be seen above, the upstream primer, KRG-8, was homologous to thebeginning 18 nucleotides of the ‘anticoding’ strand of the FBD-4encoding DNA and also carried at its 5′ end non-hybridizing DNAsequences that encoded for a Nco I site (to facilitate the ‘docking’ ofthe SOE product into the Nco I site of the expression vector, thusrecreating the ORF for the FBD(4,5)-SK fusion sequences). The upstreamprimer also contained sequence coding for a transglutaminasecross-linking site. The downstream primer KRG-9 was designed tohybridize with the end of the FBD(5) DNA sequence, but also contained atits 5′ non-hybridising end, nucleotide complementary to the first 5codons of the ORF encoding mature S. equisimilis SK (see FIG. 6). Thetemplate used for the first PCR to obtain Block I (see FIG. 20) wasFBD(4,5) cloned in pBlueScript [pSKMG400-FBD(4,5)]. The first PCR(termed PCR-I) was carried out using approx 20 ng template and 30 pmolof each primer in a 100 uL-reaction using the buffer provided byStratagene Inc., the supplier of the pfu thermostable polymerase. ThePCR employed 25 cycles with the following conditions: 94° C. for 45 sec(denaturation step) followed by 50° C. for 1 min (annealing step), and72 C for 1 mm (extension step). This was followed by an incubation for 4min at 72° C. for extension of any incomplete chains. The PCR resultedin the generation of a single species of DNA, in accordance with thesize expected from the fusion construct (368 bp), as observed by agarosegel electrophoresis; this DNA species was isolated from the gel as asmall agarose black, and subjected to further purification using theagarase treatment method, described earlier.

For obtaining the DNA Block II for the SOE reaction, only the region ofthe SK-encoding polynucleotide DNA corresponding to nucleotide 1 to 186(approx. corresponding to the first 63 amino acid residues of SK, seeFIG. 3) was amplified using pSKMG400 as template in PCR II, using theprimer set KRG 11 (upstream primer) and KRG 12 (downstream primer). Thisregion encompasses the unique Afl II site in the SK gene (see FIG. 4),The upstream primer contained non-hybridizing bases that were homologousto the last five codons of the FBD(5)-encoding DNA (viz., codons255-259), followed by a stretch of bases hybridizing to the first 27bases of the anti-sense strand of the SK-encoding ORF (see FIG. 3). Thedownstream primer contained sequences hybridizing with the stretch ofDNA encoding for residues 55-63 of SK containing the Afl II restrictionsite so that the SOE product could be docked back into the full-lengthSK-encoding polynucleotide segment contained in the vector used for theexpression of the hybrid gene (see FIG. 21 a). The PCR was carried outessentially as described for PCR I, above, except that 90 ng of templatewas chosen and the cycling conditions selected had a lower annealingtemp. (43° C.) dictated by a relatively lowered T_(m) of one of theprimers. The PCR gave a single DNA band of the expected size (201 bp) onagarose gel electrophoresis, which was isolated and purified as for PCRI product (Block 1). Splice overlap extension reaction (PCR III) wasthen carried out to obtain the hybrid DNA between the FBD and SK ORFs.In this reaction, approx. equivalent amounts of the purified DNAs fromPCR I and PCR II were mixed together (representing approx. one-fifteenthof the amplified DNA obtained from PCRs I and II) in a 100-uL reaction.To bring about optimal and specific annealing between the hybridizingareas of the two partially complementary strands from the FBD(4,5) andSK 1-63 DNA blocks (see. FIG. 20) (Phase 1), the reaction was firstcarried out in the absence of any other primers, using pfu DNApolymerase and the buffer specified by its supplier, employing thefollowing conditions: 98° C. for 2 min, slow temperature decrease (i.e.ramp of 4 min) to 50° C., maintenance at 50° C. for 1 min, followed by 3min at 65° C. A hot start was used for the initiation of the PCR (i.e.the DNA polymerase was added into the reaction after all othercomponents had been added and thermally equilibrated to the highesttemperature in the cycle). A total of 10 cycles were carried out first(Phase I), to allow formation of overlapped extended products. In thesecond phase, primers KRG 8 and KRG 12 were added under hot startconditions, and another 25 cycles were given at the following cyclingconditions: 94° C. for 1 min (denaturation step), 40° C. for 1 min(annealing), followed by extension at 72° C. for 1 min to amplify thefusion products. Finally, after 10 min at 72° C., an aliquot from thePCR was analysed by agarose gel electrophoresis. It showed the cleanappearance of the expected hybrid product (539 bp) with the absence ofany other background bands. This was isolated from the agarose gel,purified and then kinased with T4 phage polynucleotide kinase bystandard protocols. The kinased (i.e. 5-phosphorylated) product was thenblunt-end cloned at the Eco RV site of pBlueScript. Clones containingthe SOE product were selected by restriction enzyme digestion to isolatethe inserts and measuring their size by agarose gel electrophoresis. Twopositive clones were the sequenced to confirm the identity of their DNAinserts as well as the absence of any mutations (see FIG. 21 b). Nco Iand Afl II digestion of one of these two clones, the Nco I-Afl IIfragment carrying the FBD4(,5)-SK hybrid polynucleotide cassette wasligated with Nco I-Afl II digested SK-expression plasmid (pET(23d)-SK)and transformation of E. coli XL-Blue cells was carried out to obtainthe hybrid FBD(4,5)-SK ORF in this vector (FIG. 21 a). The resultantplasmid pET23(d)-FBD(4,5)-SK has been deposited with MTCC underaccession No. BPL 0015. This plasmid construct was transformed into E.coli BL-21 cells to monitor expression of the hybrid FBD-SK constructfrom the T7 RNA polymerase promoter-based vector, as described before.The SDS-PAGE gels showed the expression intracellularly of a proteinwith the expected MW (approx. 57 kD) at a level of around 20 percent oftotal intracellular, soluble protein.

Example 6

In-frame fusion of DNA segments encoding for FBD segments 4 and 5 theends of the DNA ORF encoding for SK, and cloning and expression of thehybrid polynucleotide-construct so formed, FBD(4,5)-SK-FBD(4,5), in E.coli.

The steps involved in the construction of a SK-FBD polynucleotide hybridwherein the FBD(4,5) domains were fused in-frame simultaneously at bothends (i.e. the N- and C-termini) of the SK-encoding ORF [i.e.FBD(4,5)-SK-FBD(4,5)] are shown schematically in FIG. 22 a. It is basedon the cloning of the FBD(4,5)-SK(1-57 residues) cassette obtained frompBlueScript-FBD(4,5)-SK vector, described in Example 5 (above) intopET423d)SK-FBD(4,5) at the beginning of the ORF for SK. Approximately 5ug each of pET(23 d)SK-FBD(4,5) and pBlueScriptFBD(4,5)-SK plasmid DNAwere digested with Afl II and Nco I restriction endonucleases and thedigests were electrophoresed on 1.2% agarose gels alongwith standard DNAsize markers by standard methods. In each case, two fragments wereobserved, corresponding to the vector DNAs (expected size, 5012 bp)devoid of the Nco I-Afl II fragment, and the latter fragment [approx.520 bp in the case of pBlueScriptFBD(4,5)-SK and 140 bp in case ofpET-23d)SK-FBD(4,5)] released from the parent vectors as a result of thedouble-digestion (see FIG. 22 a showing the Afl II and Nco I sites inthe two vectors). The NcoI-Afl II fragment from pBlueScript FBD(4,5)-SKcontaining the FBD(4,5)-SK (1-57 residues) cassette, to be used asinset, and the NcoI-AflII digested vector DNA from pET-23d)SK-FBD(4,5)were isolated from the agarose gel and purified. Both fragments werethen subjected to ligation using T4 DNA ligase under standard conditionsusing a molar ratio of 1:2 of vector to insert DNA. The ligation mixturewas then transformed into electro-competent E. coli XL-1 Blue cells.Positive clones, with both ends of SK fused with the FBD(4,5) domains,were selected on the basis of difference in size from the parentvectors, as well as their ability to yield the expected fragment(containing FBD sequences at both ends of the insert) after digestionwith Nco I and Bam HI enzymes (see FIG. 22 a). The veracity of theconstructs was then established by subjecting one of the selected clonesto automated DNA sequencing using Sanger's di-deoxy method to sequencethe entire hybrid ORF (see FIG. 22 b). This demonstrated that theconstruct had the expected design and sequence, with one ‘set’ of FBD4,5domain fused at the beginning of the SK-encoding polynucleotide, andanother at its end (i.e. after DNA encoding for residue 383). Thisplasmid construct has been designated pET23(d)FBD(4,5)-SK-FBD(45)], anddeposited with MTCC (Accession No. BPL 0016 in host E. coli XL-Blue). Itwas also used to transform E. coli strain BL-21 electro-competent cells,in order to express the FBD-SK-FBD hybrid construct intracellularly inE. coli. The hybrid gene was then expressed in E. coli intracellularlyafter induction with IPTG exactly as described earlier, and the celllysates analysed by SDS-PAGE. These showed the expression of apolypeptide of approx. 65 kD as expected from the incorporation of thetwo FBD segments at each ends of the SK (1-383) gene. The level ofexpression of this protein was observed to be approx. 20-25 percent ofthe total soluble protein fraction.

Example 7

Purification of various chimeric constructs formed between SK and FBDsafter expression in E. coli and refolding, and testing of their affinityfor human fibrin.

Fifty ml LB-Amp (containing 100 ug/ml of ampicillin per ml) were seededwith E. coli cells harbouring either pET23(d)-SKFBD(4,5),pET23(d)-SKFBD(1,2), pET-23(d)FBD(4,5)-SK orpET23(d)-FBD(4,5)-SK-FBD(4,5-) plasmid constructs in separate flasks(150 ml capacity). The inoculation was done from the respective glycerolstocks, and the culture was incubated at 37° C. for 12 h on a rotaryshaker (200 r.p.m.). This pre-inoculum was used to seed fresh LB-amp at5% (v/v) level (one litre total for each type of E. coli BL-21 cellsharbouring one of the different plasmid constructs described above with500 ml medium per 2 liter conical flaske), and the cultures shaken asabove at 37° C. for approx. 2 h 30 min, a which time the OD600 of thecultures bad reached a value of 1.0-1.1. The expression of chimericSK/FBD polypeptides in the cultures were then induced by the addition ofIPTG to 1 mM, and continuing further the incubation for another 3 h.Cells from all four cultures were then harvested by high-speedcentrifugation (8000 g×30 min) at 4° C., and washed once with 500 mlcold ST buffer (pH 7.5). Finally, each cell pellet was suspended in cold25 ml ST buffer, pH 8.0. The wet-weight of the pellets obtained from 1liter cultures varied between 4.3-4.5 g. Each cell-suspension was thensubjected to ultra-sonication to effect cell-lysis using standardmethods. The lysates so obtained (approx. 28 ml each) were subjected tohigh-speed centrifugation at 4° C. to pellet any unlysed cells and/orcell debris. The protein conc. in the supernatants varied between 15.0to 16.0 mg/ml. These were then diluted to a final protein of 1 mg/mlusing distilled water, together with the addition of the followingadditional components (final concentrations in the diluted mix aregiven): Tris-Cl, pH 8.0, 50 mM; NaCl 150 mM; EDTA 1 mM; mixture ofreduced and oxidized glutathione 123:50 mg. To these refolding mixtures(approx. 400 ml each) were then added 30 ml (packed volume) offibrin-Sepharose beads pre-equilibrated with 50 mM Tris-Cl, pH 8.0. Themixtures were stirred at 22° C. for 16 h to effectreoxidation/refolding. The solutions were then passed through 50ml-volume axial glass columns fitted with fitted glass disks (to retainthe Sepharose-beads). The packed fibrin-Sepharose beds were then washedwith approx. 170 ml binding buffer (50 mM tris-Cl, pH 8.0, and 150 mMNaCl), followed by 100 ml of 2 M urea (in binding buffer), and finallythe fibrin-bound protein was eluted with 6 M urea (in binding buffer).All the washing/elution steps were carried out at a flow rate of −30ml/h using a peristaltic pump assembly. The chromatographic profile incase of SK-FBD(4,5) fibrin-Sepharose affinity purification, and analysisof the different fractions, are show on FIG. 23, Similar results wereobtained in case of the other SK-FBD constructs. A total of 3.8 mg ofprotein was eluted alongwith the 6 M urea-wash in the case ofSK-FBD(4,5), whereas for SK-FBD(1,2) approx. 4 mg, for FBD(4,5)-SK 3.5mg, and for FBD(4,5)-SK-FBD(4,5) 6.2 mg of protein was obtained at the 6M urea elution step. In case of the SK control, no protein was found toelute alongwith the 6 M urea. The removal of the urea, and concentrationof the protein, was effected by ultrafiltration through 10 kD cut-offMEMBRANES. Aliquots of each of the four fibrin-specific SK-FBD chimericproducts were then subjected to SDS-PAGE analysis on 10 percentacrylamide gels with and without reduction with beta-mercaptoethanol todetermine their relative purities as well as monomeric/polymericcharacter. Standard molecular weight marker proteins as well as purenative SK from S. equismilis were also run on the same gels. TheSDS-PAGE analysis, either in the presence or absence of reducing agent,showed all of the fibrin-Sepharose eluted chimeric product to beessentially pure and monomeric; in all cases, a single prominent bandstained with Coomassie Blue and very few faint background bands werevisible (not more than 2-5% cumulatively). The MWs of the refoldedproteins were completely in accord with those expected from theoreticalconsiderations i.e. extrapolated from the MW of individual domains ofFBD, the SK portion, and linker sequence, if present. The SK-FBD(4,5)and SK-FBD(1,2) bands moved with the same mobility on SDS-PAGE, with anapparent MW of around 55 kD; however, the FED-(4,5)-SK construct showeda slightly lowered mobility as compared to either SK-FBD(4,5) orSK-FBD(1,2). This was in accord with the fact that whereas the formertwo hybrid constructs contained approx. 31 amino acid residues' deletionat the C-terminal end of the SK moeity of the hybrid, the FBD(4,5)SKconstruct had full-length SK integrated in its design (see Examples,above). The FBD(4,5S)SK-FBD(4,5) construct, containing four FBDsalongwith SK, moved with a MW corresponding to 60 kD on SDS-PAGE. In theabsence of beta-mercaptoethanol, the MW's calculated for all fourhybrids were approximately the same as observed in the presence ofbeta-mercaptoethanol, indicating that the constructs obtained afterrefolding and binding with fibrin-Sepharose contained essentiallymonomeric forms of the polypeptides.

The specific activities of the purified proteins for PG activation, asdetermined by the chromogenic assay were: 2.2.×10⁴ I.U./mg forSK-FBD(4,5), 1.8×10⁴ I.U./mg for SK-FBD(1,2). 4×10⁴ I.U./mg in case ofFBD(4,5)-SK and 5×10² I.U./mg for FBD(4,5)-SK-FBD(4,5), respectively.Under the same conditions, native S. equisimilis SK, or SK purified fromE. coli (Met-SK) as described in Example 2, above, showed a much higheractivity (.about.1.0×10⁵ I.U./mg). The reason for the apparently loweredspecific activities in case of the chimeric proteins was revealed whenthese were assayed by a single-phase, continuous spectrophotometricassay by directly determining their rates (slopes) for HPG activation bystandard methods (Wohl, R. C., Summaria, L., and Robbins, K. C., 1980,J. Biol. Chem. 255:2005). These assays revealed that whereas native SKor E. coli-expressed Met-SK did Dot display an appreciable lag in theprogress curves obtained for the PG activation reactions (less than 1min), all of the hybrid proteins displayed significant initial periodsin their PG activation profiles (varying from 7 to 25 min depending onthe construct) wherein little or no plasmin formation occurred. However,after the initial lags, the PG activation proceeded with a high rate,generating slopes closely similar to those obtained with native SK (seebelow).

Example 8

Functional characterization of the chimeric proteins in terms of theiraltered kinetics of plasminog n activation and fibrin clot dissolution.

The proteins prepared in Example 7, above, as well as native and Met-SR(as controls) were examined with respect to their PG activationkinetics. This essentially entailed the study of the time-course of PGactivation by the various SK/FBD chimeras and the determination of theirsteady-state kinetic constants for PG activation. A one-stage assaymethod was used to measure the activation of HPG; reference in thiscontext may be made to several publications in the literature e.g., Shi,G. Y., Chang, B. I., Chen, S. M., Wu, D. H. and Wu, H. L., 1994,Biochem. J. 304:235; Wu, H. L., Shi, G. Y., and Bender, M. L., 1987,Proc. Natl. Acad. Sci. 84: 8292; Wohl, R. C., Summaria, L., and Robbins,K. C., 1980, J. Biol. Chem. 255:2005; Nihalani D., Raghzva, G. P. S.,Sahni, G., 1997, Prot. Sci. 6:1284). Briefly, it involved the additionof the activator proteins to be studied in a small aliquot (˜5 ul) into100 ul-volume microcuvette containing 1 uM of HPG in assay buffer (50 mMTris-Cl buffer, pH 7:5, containing 0.5 mM chromogenic peptide substrateand 0.1 M NaCl). The protein aliquots were added after addition of allother components into the cuvette and bringing the spectrophotometricabsorbance to zero. The change in absorbance at 405 nt was then measuredas a function of time in a Shimadza UV-160 model spectrophotometer.While SK showed a rapid PG activation kinetics, the kinetics for SK-FBDchimeric protein displayed a characteristic lag, or delay, in theinitial phase of the rate of PG activation that was clearly differentfrom the rates seen with SK. This property viz., initial delay in HPGactivation, as well as its magnitude, was largely independent of theamount of the chimeric protein employed in the assay, as well as theconcentration of HPG in the reaction. Another notable feature was thatthe lag-times associated with the different chimeric proteins under thesame conditions. In the case of SK-FBD(1,2) and SK-FBD(4,5) the lagperiod corresponded to 10-12 min, for FBD(4,5)-SK 7-8 min, and 20-25 minin case of FBD(4,5)-SK-FBD(4,5). Under the same conditions; (−1 uM HPG,1-2 nM of protein), native SK or Met-SK displayed very little lag period(i.e. less than 1 min duration) during PG activation.

The mechanism of the initial lag in the various SK-FBD chimeras wasinvestigated by examining the SDS-PAGE profiles of various aliquotswithdrawn from plasminogen activation reactions withdrawn at differenttime-intervals after the mixing of SK or SK-FBD chimeric protein withhuman PG. These showed that the appearance of rapid PG activationfollowing the lag period closely coincided with the cleavage of the FBDportion from the rest of the molecule (SK portion) as evidenced by areduction of the molecular weight of the hybrid. That the proteolysiswas mediated by trace amounts of plasmin in the system was evident bythe observation that either removal of trace plasmin by passage of thehuman PG through soybean trypsin inhibitor agarose (a material thatselectively binds plasmin and does not bind plasminogen) led to veryhigh periods of lag for all of the hybrid proteins [viz., from 10-12 minto approx. 25 min for SK-FBD (1,2), SK-FBD(4,5, and FBD(4,5)-SK; toapprox. 35 min for FBD(4,5)SK-FBD(4,5) from an initial value of approx.20 min]. Alternatively, the addition of small quantities of preformedhuman plasmin into the PG activation reactions (made by the conversionof PG to plasmin with agarose-immobilized urokinase) considerablyreduced the lag periods associated with the different SK-FBD chimeras.

To determine the steady-state kinetic parameters for HPG activation ofthe activated forms of the hybrids, fixed amounts of SK or SK-FBDchimeric protein (1 nM) were added to the assay buffer containingvarious concentrations of HPG (ranging from 0.035 to 2.0 uM) in the 100uL assay micro-cuvette as described above. The change in absorbance(representing velocity, v) was then measured spectrophotometrically at405 nM for a period of 30 min at 22° C. All determinations were done intriplicates and their averages taken for analysis. The kineticparameters for HPG activation were then calculated (using the linearportion of the progress curves) from inverse, Lineweaver-Burke plotsusing stan procedures (Wohl, R. C., Summaria, L., and Robbins, K. C.,1990., J. Biol. Chem. 255: 2,005), wherein the 1/v value is plotted onthe ordinate axis and 1/S value is plotted on the abscissa, Srepresenting the (varying) concentration of substrate (HPG) employed forthe reaction/s. From these plots, the K_(m) for HPG (K_(plg)) andmaximal velocities (at saturating HPG concentrations) were determined(set forth in the following Table).

These data clearly show that once fully activated after completion ofthe initial lag, all the chimeric constructs became significantly activein terms of their PG activator abilities in comparison to SK.

TABLE 1 Steady-state kinetic parameters for HPG activation by SK andSK-FBD hybrid proteins* Activator ^(k)plg Lag protein (μM) Maximalactivity# (min) nSK 0.14 ± 0.02 100.0 1.0 Met-SK 0.18 ± 0.01 95.5 ± 5  2.0 SK-FBD(4, 5) 0.15 ± 0.02 52 ± 4 10.0 SK-FBD(1, 2) 0.18 ± 0.03 58 ± 510.5 FBD(4, 5)-SK 0.16 ± 0.02 65 ± 4 8.0 FBD(4, 5)-SK-FBD(4, 5) 0.20 ±0.03 45 ± 4 18.0 *The parameters were calculated from the linear phasesof the reaction progress curves after the abolishment of the lag phases.#Expressed relative to the activity of native SK from Streptococcus sp.(ATCC 12449), taken as 100 percent.

In a separate series of experiments, the rates of proteolyticdissolution of radiolabelled fibrin clots in vitro was examined to testwhether, like native SK. The SK-FBD chimeric proteins could alsoefficiently break down fibrin to soluble products, a fundamentalbiological property of all thrombolytic agents, and also to examine ifthe altered PG activation kinetics observed with synthetic peptidesubstrate, described above (i.e. slow initial rates, followed by ratesclose to those observed for native SK) were also reflected at the levelof clot lysis.

Radioactive fibrin clots were first prepared-by mixing 400 ul of coldfibrinogen (2.5 mg/ml stock) with 50 ul of ¹²⁵I-labelled fibrinogencontaining 9×10⁵ cpm (specific activity 7.2×10⁵ cpm/ug protein) andadding to a solution (150 ul) containing 100 ug HPG and 0.25 N.I.H.units of thrombin (Sigma). All solutions were made in 0.1 M citratephosphate buffer, pH 7.5, containing 0.8 percent BSA (BSA-citratebuffer). The final volume of the clotting reaction was adjusted to atotal volume of 1 ml with BSA-citrate buffer. The clot was formed byincubating the mixture in a glass tube at 37° C. for 2 min. The clot wasthen washed thrice with 2 ml of TN buffer (50 mM Tris-Cl buffer, pH 7.5,containing 38 mM NaCl and 0.01 percent Tween-80) for 3 min at 37° C.When required non-radioactive fibrin clots were prepared exactly asdescribed above but omitting the inclusion of ¹²⁵I-labelled fibrinogenfrom the clotting mixture. The effect of the thrombolytic agent (nativeSK or SK-FBD hybrid) was then studied in terms of release ofradioactivity from the clot kept either in a plasma milieu or inpresence of excess human fibrinogen as described below.

Clot lysis of preformed fibrin clots suspended in human plasma wascarried out by suspending ¹²⁵I-labelled and extensively washed clots in2 ml citrated human plasma, prewarmed at 37° C., and adding differentamounts of either SK or a given SK-FBD hybrid protein. The reactiontubes were rotated slowly at 37° C. in a water bath and 0.1 ml aliquotsof the soluble fraction were removed at regular intervals to measure the¹²⁵I-fibrin degradation products released by measuring the amount ofradioactivity using a gamma counter. The total radioactivity in eachclot was determined by measuring the radioactivity of the respectivetube before withdrawing any aliquot prior to the addition ofthrombolytic agent. A comparison of the dissolution kinetics ofradio-labelled fibrin clots by native SK and the various SK-FBD chimericproteins in plasma milieu also clearly showed that the lag displayed bythe latter during the PG activation assays was essentially preservedduring clot lysis also. While SK caused relatively rapid dissolution ofthe fibrin and a patterning of the dissolution reaction at or around 15min, a prolonged lag in the case the SK-FBD(4,5) hybrid protein (approx.10 min) was evident at the same protein concentrating (representativedata for those two proteins are shown in FIG. 24). In the case of theother hybrids; the lag-times in plasma were essentially as seen with PGactivation assays viz., 10 min for SK, FBD(1,2), 8 min for FBD(4,5)-SK,and 18 min for FBD(4,5)-SK-FBI(4,5).

Clot lysis in the presence of an excess of human fibrinogen was alsocarried out by measuring the rate of dissolution of radio-labeled fibrinclot by SK or SK-FBD protein in the presence of various concentrationsof human fibrinogen (1-4 mg/ml) and 100 nM of either SK or SK-FBD hybridprotein. Clot lysis was also performed in the presence of fixedfibrinogen concentration (2 mg/ml) but employing differentconcentrations of SK/SK-FBD protein (ranging from 50 to 200 mM). Thereactions were incubated at 37° C. in a water bath with gentle shaking,and the release of ¹²⁵I-fibrin degradation products as a function oftime was measured in the supernatant, as described above. All of thehybrid proteins were able, like SK, to dissolve the fibrin clots in adose-dependent manner; however, there was a distinct lag in the case ofthe SK-FBD hybrids closely similar to that seen with clot lysis inplasma milieu. The lag period varied with construct design viz in caseof SK an absence of any appreciable lag was observed (less than 2 min).The lag times for SK-FBD(4,5) and SK-FBD(1,2) were 10-11 min; forFBD(4,5)SK 7-8 min; and 18-20 min for FBD(4,5)-SK-FBD(4,5).

ADVANTAGES OF THE INVENTION

The advantage of the present invention lies in its disclosure of thedesign of structurally defined SK-FBD chimeric polynucleotide DNAs inwhich the translational in-frame fusion of the DNAs encoding SK, or itsmodified forms, and those for the minimally essential human Fibronectingene that are capable of possessing significant fibrin affinity on theirown, such as those FBDs that possess independent fibrin bindingcapability (e.g., “finger” domains 4 and 5 of human fibronectin) hasbeen carried out in such a manner that the polypeptide/s expressed fromthese polynucleotide constructs possess fibrin affinity (which SK, onits own, does not possess) together with a delayed PG activationkinetics (unlike SK which show an immediate activation of PG).

The simultaneous presence of the afore-mentioned properties in the samePG activator confers distinct advantages into the resultant proteins.Soon after injection into the body, whilst the chimeric PG activatorproteins are still in an inactive or partially active-state, they willbind to the pathological fibrin clot during their sojourn through thevascular system in an inactive/partially active state. However, after aninitial lag, these will become fully activated in the immediate vicinityof the clot, thereby obviating the systemic PG activation coincidentwith natural SK administration. Whilst the former property would beexpected to confer on the thrombolytic agent/s an ability to targetitself to the immediate locale of the pathological clot and thus helpbuild up therapeutically effective concentrations of the activatortherein, the initially slowed kinetics of PG activation would result inan overall diminished generation of free plasmin in the circulationprior to their localization to the site of circulatory impedance inducedby the pathological fibrin clot. The net result shall be a continued andmore efficient fibrinolysis at the target sustained by considerablylowered therapeutically effective dosages of the thrombolytic agent.

1. A nucleic acid molecule comprising a nucleotide sequence encoding ahybrid polypeptide, said hybrid polypeptide comprising at least onefunctional fragment of streptokinase (SK) and fibrin binding domains 4and 5, or fibrin binding domains 1 and 2 of human fibronectin, saidfunctional fragment of SK and said fibrin binding domains beingconnected via a flexible connecting oligopeptide, said SK fragmentlacking plasminogen activation function unless said fibrin bindingdomains are bound to fibrin.
 2. The nucleic acid of claim 1, whereinsaid fibrin binding domains of human fibronectin are domains 4 and
 5. 3.A vector of comprising the nucleic acid of claim
 2. 4. A host cellcomprising the vector of claim
 3. 5. The host cell of claim 4, whereinthe host cell is selected from the group comprising E. coli, Bacillusspp., yeast, fungus, plant and animal.
 6. A method of producing a hybridpolypeptide comprising culturing the host cell of claim 4 underconditions suitable for protein expression and isolating the expressedhybrid protein.
 7. The nucleic acid of claim 1, wherein said fibrinbinding domains of human fibronectin are domains 1 and
 2. 8. A vector ofcomprising the nucleic acid of claim
 6. 9. A host cell comprising thevector of claim
 7. 10. The host cell of claim 8, wherein the host cellis selected from the group comprising E. coli, Bacillus spp., yeast,fungus, plant and animal.
 11. A method of producing a hybrid polypeptidecomprising culturing the host cell of claim 8 under conditions suitablefor protein expression and isolating the expressed hybrid protein.